Cutting Edge Genetics: CRISPR/Cas9 Editing of Plant Genomes

Cutting Edge Genetics: CRISPR/Cas9 Editing of Plant Genomes Abstract The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system is a genome editing technology transforming the field of plant biology by virtue of the system’s efficiency and specificity. The system has quickly evolved for many diverse applications including multiplex gene mutation, gene replacement and transcriptional control. As CRISPR/Cas9 is increasingly applied to plants, it is becoming clear that each component of the system can be modified to improve editing results. This review aims to highlight common considerations and options when conducting CRISPR/Cas9 experiments. Introduction The ability to manipulate gene function has been an indispensable tool for basic plant research and for the generation of improved crop varieties. Whether in the lab or in the field, the ability to modulate gene function would ideally employ technology that is specific, efficient and heritable. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) genome editing technology provides such a tool, enabling revolutionary advances in both arenas of plant biology. CRISPR/Cas systems are bacterial adaptable immune systems against foreign DNA sources such as bacteriophages or plasmids (Wiedenheft et al. 2012). CRISPR/Cas immunity is mediated by RNA-guided nucleases, which target invading phage genomes for degradation in a sequence-specific manner, conferring immunity. There are five distinct CRISPR/Cas systems identified in bacteria which differ in their gene composition and ability to cleave phage DNA, RNA or both. These systems have been co-opted for genome editing in heterologous species for their ability to confer sequence-specific targeting and cleavage of DNA, or RNA, targets. The Type II mechanism is the most commonly used in genome editing for heterologous systems because it requires only one Cas protein, Cas9, for DNA targeting and cleavage (Deltcheva et al. 2011). CRISPR/Cas immunity in bacteria consists of an adaptation phase, an expression (or biogenesis) phase and an interference phase (Jiang and Doudna 2016). During the adaptation phase, a DNA record of an attempted phage infection is created when an invading phage genome is cleaved by endonucleases into short DNA sequences, which are then stored in the bacterial genome in so-called CRISPR DNA arrays (Barrangou et al. 2007). The CRISPR DNA array consists of short conserved sequence repeats separated by short variable sequences of spacer DNA, each corresponding to a unique captured foreign DNA sequence. During the expression phase, the entire CRISPR array is transcribed and processed into small CRISPR RNAs (cRNAs), consisting of one captured spacer sequence and one repeat sequence each (Jiang and Doudna 2016). A cRNA hybridizes with a second RNA, the trans-activating CRISPR RNA (tracrRNA), to form a small hybrid RNA molecule, the crRNA (Deltcheva et al. 2011). During the interference phase, the mature crRNA hybrid binds the Cas9 endonuclease and is recruited to invading phage DNA (Jiang and Doudna 2016). Binding of the Cas nuclease–crRNA complex is dependent on two factors: (i) complementary base pairing between the spacer sequences on the crRNA and corresponding phage DNA ‘protospacer’ sequence; and (ii) a protospacer adjacent motif (PAM), a short nucleotide consensus sequence that flanks the protospacer sequence and is critical for Cas9 nuclease binding (Gasiunas et al. 2012, Jinek et al. 2012). Streptococcus pyogenes Cas9 (SpCas9) cleaves target DNA sites flanked by a PAM sequence conforming to a 5'-NGG-3' consensus immediately following the 3' end of the protospacer target, although alternative variant PAMs will be cleaved by Cas9 at lower efficiencies (Anders et al. 2016). The requirement for the PAM sequence in target binding by the Cas9–crRNA insures that the complex will not bind and cleave spacer sequences in the bacterial genomic CRISPR array, which lacks PAM sites. Pioneering work from the labs of Charpentier and Doudna showed that the ability to target and cleave DNA in a sequence-specific manner could be reduced in vitro down to just Cas9 and a single synthetic RNA which fuses the tracrRNA and crRNA together into a single guide RNA (gRNA) (Jinek et al. 2012). In this work, they demonstrated that from these simple principles gRNAs could be designed that target protospacers adjacent to PAM sites in a GFP (green fluorescent protein) gene, and Cas9 would then cleave the double-stranded GFP DNA. Subsequent work from the Church and Zhang groups extended these principles to human cells, showing that you could exploit these properties to target Cas9–gRNA complexes to DNA in a sequence-specific manner (Cong et al. 2013, MaLi et al. 2013). Eukaryotic gene function could be disrupted by creating double-stranded DNA breaks (DSBs) repaired incorrectly by non-homologous end-joining (NHEJ) DNA repair mechanisms, resulting in nucleotide insertions and/or deletions (indels) that generate frameshift mutations. In addition to donor DNA repair templates, Cas9-mediated DNA breaks could be exploited to knock-in donor DNA sequences by the homology-dependent repair (HDR) DNA repair pathway, effectively replacing endogenous gene sequences. Excitingly, distinct gRNAs targeting different genomic sites could be combined simultaneously to multiplex edit different genome sites at once. Together these three papers set off the current revolution in genome engineering, which quickly extended to plants. Central to any use of CRISPR systems in genome editing is the desire to edit target sites efficiently and specifically. As the use of Cas9 has expanded, different strategies have emerged that impact experimental success. Here we compare and discuss Cas9 approaches in plants, using current literature and experiences in our lab, and discuss caveats and considerations with this technology. A summary of the optimizable parameters of the CRISPR/Cas9 system for plants discussed in the present review can be viewed in Table 1. Table 1 Optimizable parameters in the design of CRISPR/Cas9 systems for plants Feature Optimization Options Reference(s) gRNA Design GC content Ma et al. 2015, Pan et al. 2016, Zhang et al. 2014 Target accessibility Kuscu et al. 2014, Wu et al. 2014 Number of mismatches Xing et al. 2014 Spacer length Osakabe et al. 2016 Delivery Systems Agrobacterium- mediated delivery Ma et al. 2015 Biolistic delivery Shan et al. 2013, Jacobs et al. 2015, Svitashev et al. 2015 RNP delivery Woo et al. 2015, Liang et al. 2017, Svitashev 2016 TECCDNA/TECCRNA delivery Zhang et al. 2016, Liang et al. 2017 Viral delivery Čermák et al. 2015, Gil-Humanes et al. 2017 Cas9 expression systems CaMV 35s promoter (for transient assays, not recommended for stable heritable events) Feng et al. 2013, Jiang et al. 2013, Shan et al. 2013 Species appropriate Ubiquitin promoter Miao et al. 2013, Wang et al. 2014, Peterson et al. 2016 Egg cell-specific (EC1.2) promoter Wang et al. 2015 SPL promoter Mao et al. 2016 LAT52 promoter Mao et al. 2016 DDT45 promoter Mao et al. 2016 Meiosis-specific (MGE1/MGE2/MGE3) promoter Eid et al. 2016 Cas9 modifications Codon optimization Fauser et al. 2014, Gao et al. 2015a, Jiang et al. 2013b, Miao et al. 2013, Shan et al. 2013 Paired nickases Fauser et al. 2014, Mikami et al. 2016, Schiml et al. 2014 dCas9 Lowder et al. 2015, Piatek et al. 2015 Heat shock treatment LeBlanc et al. 2018 Alternative endonuclease varieties SaCas9 Zhang et al. 2017, Steinert et al. 2015 St1Cas9 Steinert et al. 2015 SpCas9-HF1 Kleinstiver et al. 2016 eSpCas9(1.1) Slaymaker et al. 2016 HypaCas9 J.S. Chen et al. 2017 Cpf1 Xu et al. 2017, Kim et al. 2017 Cas13a Abudayyeh et al. 2017 Feature Optimization Options Reference(s) gRNA Design GC content Ma et al. 2015, Pan et al. 2016, Zhang et al. 2014 Target accessibility Kuscu et al. 2014, Wu et al. 2014 Number of mismatches Xing et al. 2014 Spacer length Osakabe et al. 2016 Delivery Systems Agrobacterium- mediated delivery Ma et al. 2015 Biolistic delivery Shan et al. 2013, Jacobs et al. 2015, Svitashev et al. 2015 RNP delivery Woo et al. 2015, Liang et al. 2017, Svitashev 2016 TECCDNA/TECCRNA delivery Zhang et al. 2016, Liang et al. 2017 Viral delivery Čermák et al. 2015, Gil-Humanes et al. 2017 Cas9 expression systems CaMV 35s promoter (for transient assays, not recommended for stable heritable events) Feng et al. 2013, Jiang et al. 2013, Shan et al. 2013 Species appropriate Ubiquitin promoter Miao et al. 2013, Wang et al. 2014, Peterson et al. 2016 Egg cell-specific (EC1.2) promoter Wang et al. 2015 SPL promoter Mao et al. 2016 LAT52 promoter Mao et al. 2016 DDT45 promoter Mao et al. 2016 Meiosis-specific (MGE1/MGE2/MGE3) promoter Eid et al. 2016 Cas9 modifications Codon optimization Fauser et al. 2014, Gao et al. 2015a, Jiang et al. 2013b, Miao et al. 2013, Shan et al. 2013 Paired nickases Fauser et al. 2014, Mikami et al. 2016, Schiml et al. 2014 dCas9 Lowder et al. 2015, Piatek et al. 2015 Heat shock treatment LeBlanc et al. 2018 Alternative endonuclease varieties SaCas9 Zhang et al. 2017, Steinert et al. 2015 St1Cas9 Steinert et al. 2015 SpCas9-HF1 Kleinstiver et al. 2016 eSpCas9(1.1) Slaymaker et al. 2016 HypaCas9 J.S. Chen et al. 2017 Cpf1 Xu et al. 2017, Kim et al. 2017 Cas13a Abudayyeh et al. 2017 Table 1 Optimizable parameters in the design of CRISPR/Cas9 systems for plants Feature Optimization Options Reference(s) gRNA Design GC content Ma et al. 2015, Pan et al. 2016, Zhang et al. 2014 Target accessibility Kuscu et al. 2014, Wu et al. 2014 Number of mismatches Xing et al. 2014 Spacer length Osakabe et al. 2016 Delivery Systems Agrobacterium- mediated delivery Ma et al. 2015 Biolistic delivery Shan et al. 2013, Jacobs et al. 2015, Svitashev et al. 2015 RNP delivery Woo et al. 2015, Liang et al. 2017, Svitashev 2016 TECCDNA/TECCRNA delivery Zhang et al. 2016, Liang et al. 2017 Viral delivery Čermák et al. 2015, Gil-Humanes et al. 2017 Cas9 expression systems CaMV 35s promoter (for transient assays, not recommended for stable heritable events) Feng et al. 2013, Jiang et al. 2013, Shan et al. 2013 Species appropriate Ubiquitin promoter Miao et al. 2013, Wang et al. 2014, Peterson et al. 2016 Egg cell-specific (EC1.2) promoter Wang et al. 2015 SPL promoter Mao et al. 2016 LAT52 promoter Mao et al. 2016 DDT45 promoter Mao et al. 2016 Meiosis-specific (MGE1/MGE2/MGE3) promoter Eid et al. 2016 Cas9 modifications Codon optimization Fauser et al. 2014, Gao et al. 2015a, Jiang et al. 2013b, Miao et al. 2013, Shan et al. 2013 Paired nickases Fauser et al. 2014, Mikami et al. 2016, Schiml et al. 2014 dCas9 Lowder et al. 2015, Piatek et al. 2015 Heat shock treatment LeBlanc et al. 2018 Alternative endonuclease varieties SaCas9 Zhang et al. 2017, Steinert et al. 2015 St1Cas9 Steinert et al. 2015 SpCas9-HF1 Kleinstiver et al. 2016 eSpCas9(1.1) Slaymaker et al. 2016 HypaCas9 J.S. Chen et al. 2017 Cpf1 Xu et al. 2017, Kim et al. 2017 Cas13a Abudayyeh et al. 2017 Feature Optimization Options Reference(s) gRNA Design GC content Ma et al. 2015, Pan et al. 2016, Zhang et al. 2014 Target accessibility Kuscu et al. 2014, Wu et al. 2014 Number of mismatches Xing et al. 2014 Spacer length Osakabe et al. 2016 Delivery Systems Agrobacterium- mediated delivery Ma et al. 2015 Biolistic delivery Shan et al. 2013, Jacobs et al. 2015, Svitashev et al. 2015 RNP delivery Woo et al. 2015, Liang et al. 2017, Svitashev 2016 TECCDNA/TECCRNA delivery Zhang et al. 2016, Liang et al. 2017 Viral delivery Čermák et al. 2015, Gil-Humanes et al. 2017 Cas9 expression systems CaMV 35s promoter (for transient assays, not recommended for stable heritable events) Feng et al. 2013, Jiang et al. 2013, Shan et al. 2013 Species appropriate Ubiquitin promoter Miao et al. 2013, Wang et al. 2014, Peterson et al. 2016 Egg cell-specific (EC1.2) promoter Wang et al. 2015 SPL promoter Mao et al. 2016 LAT52 promoter Mao et al. 2016 DDT45 promoter Mao et al. 2016 Meiosis-specific (MGE1/MGE2/MGE3) promoter Eid et al. 2016 Cas9 modifications Codon optimization Fauser et al. 2014, Gao et al. 2015a, Jiang et al. 2013b, Miao et al. 2013, Shan et al. 2013 Paired nickases Fauser et al. 2014, Mikami et al. 2016, Schiml et al. 2014 dCas9 Lowder et al. 2015, Piatek et al. 2015 Heat shock treatment LeBlanc et al. 2018 Alternative endonuclease varieties SaCas9 Zhang et al. 2017, Steinert et al. 2015 St1Cas9 Steinert et al. 2015 SpCas9-HF1 Kleinstiver et al. 2016 eSpCas9(1.1) Slaymaker et al. 2016 HypaCas9 J.S. Chen et al. 2017 Cpf1 Xu et al. 2017, Kim et al. 2017 Cas13a Abudayyeh et al. 2017 Cas9 as a Genome-Editing Tool: General Considerations Target selection and the specificity of gRNAs RNA-mediated DNA homing is central to any Cas9 genome editing application, whether the goal is to knock out gene function, replace endogenous gene sequences or home Cas9 to DNA for other applications. For SpCas9, gRNA target sites are typically 19–20 nucleotides (nt) in length and are flanked on the 3' end by an NGG PAM site. Truncated gRNAs (tru-gRNAs) that are 17–18 nt, compared with the traditional 20 nt size, can increase specificity without compromising efficiency (Fu et al. 2014, Ren et al. 2014, Moreno-Mateos et al. 2015, Tsai et al. 2015). Initially tested in zebrafish, Drosophila and human cell lines, tru-gRNAs were recently used to induce mutations efficiently in Arabidopsis (Osakabe et al. 2016). Cas9 nuclease activity cuts double-stranded DNA at the –3 position relative to the PAM and is strand independent in Arabidopsis (T. Wang et al. 2014, Peterson et al. 2016). gRNAs bind to their target site by hybridizing to the antisense strand of the DNA target. Studies have shown that the GC content of the target site is a factor in editing efficiency and specificity. A GC content of 40–60% seems to be the preferred range in mammalian cell lines (Liu et al. 2016). In human cells, mismatches between gRNA and the target site are tolerated to a degree, leading to off-target Cas9 binding and cleavage events (Fu et al. 2013). The region most distal from the PAM, the tail region, is more permissive of mismatches, while the seed region (the 8–12 nt most proximal to the PAM) is critical for site recognition and also does not tolerate mismatches (Jinek et al. 2012, Liu et al. 2016) (Fig. 1). A high degree of homology within the seed region can result in off-target binding (Pattanayak et al. 2013). A favorable target site should differ from potential off-target sites by at least a few nucleotides, preferably in the seed region, to avoid the possibility of off-target editing. Online programs, such as CRISPR-P and CRISPR-Plant, can be used to design gRNAs specifically for a variety of fully sequenced plant genomes and are helpful in insuring that perfect target site matches do not exist elsewhere in the genome. Both programs use algorithms to analyze the intended target sites; CRISPR-P then identifies potential off-target sites throughout the genome (Lei et al. 2014), while CRISPR-Plant prioritizes target sites with the lowest probability of off-target editing (Xie et al. 2014). It should be noted that gRNA targeting programs identify off-targets based on extrapolated gRNA binding rules. Unbiased approaches to identify off-target events have been generated by several groups. Genome-wide, unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) works by tagging DSBs in live cells using blunt, double-stranded oligodeoxynucleotides (dsODNs) and then mapping these dsODNs via sequencing (Tsai et al. 2015). A similar method, direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (BLESS), maps DSBs using a barcode-containing biotinylated linker (Crosetto et al. 2013). Chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) using catalytically inactive Cas9 can also identify Cas9-binding sites, (Kuscu et al. 2014), but binding does not always correlate with editing (Wu et al. 2014, Tsai et al. 2015). To our knowledge, these methods have not been used to detect editing in plants, but theoretically could be applied to plant protoplasts (Shi et al. 2016). More importantly, unbiased approaches that enrich for Cas9 cleavage events have revealed that for SpCas9 there is little overlap between predicted off-target sites and true off-target sites (Tsai et al. 2015). As such there is still much to learn about target selection and much remains to be confirmed in plants. Fig. 1 View largeDownload slide Overview of the CRISPR-Cas9 system. The system consists of the gRNA (white), which guides the Cas9 nuclease to the genomic target site (pink). The genomic target region (purple indicator) is composed of 20 bp that are homologous to the gRNA (white) and a PAM sequence (green indicator). The tail region (orange indicator) of the genomic target, which is most distal from the PAM motif, is more permissive of mismatches, while the 8–12 nt seed region (blue indicator) most proximal to the PAM is critical for site recognition of the CRISPR/Cas9 complex. Cleavage occurs 3bp 5’ of the PAM sequence. Fig. 1 View largeDownload slide Overview of the CRISPR-Cas9 system. The system consists of the gRNA (white), which guides the Cas9 nuclease to the genomic target site (pink). The genomic target region (purple indicator) is composed of 20 bp that are homologous to the gRNA (white) and a PAM sequence (green indicator). The tail region (orange indicator) of the genomic target, which is most distal from the PAM motif, is more permissive of mismatches, while the 8–12 nt seed region (blue indicator) most proximal to the PAM is critical for site recognition of the CRISPR/Cas9 complex. Cleavage occurs 3bp 5’ of the PAM sequence. Despite these concerns, there is little actual off-target editing in Arabidopsis, tomato, maize, rice and tobacco when using SpCas9 compared with animals (Nekrasov et al. 2013, Feng et al. 2014, Gao et al. 2015, Svitashev et al. 2015, Woo et al. 2015, Ishizaki 2016, Pan et al. 2016, Peterson et al. 2016, Tang et al. 2016). In the most comprehensive study to date in Arabidopsis, pooled plants expressing 14 gRNAs targeting different genomic sites were analyzed by deep sequencing. On-target indel rates ranged from 33% to 92% of sequencing reads, but no off-target editing events were found elsewhere in the genome at predicted or other sites, confirming smaller scale studies (Peterson et al. 2016). In tomato, no off-target mutation events were found during editing of pathogen-related genes (Nekrasov et al. 2017), or when engineering tomato quantitative trait variation via targeted deletion of cis-regulatory elements (Rodríguez-Leal et al. 2017). Outside of Arabidopsis, the use of spCas9 has led to off-target events at low frequencies in some cases. Whole-genome sequencing experiments screening for off-target editing in rice revealed no large-scale off-target editing mutations in one study (Zhang et al. 2014), but low levels of off-target editing were detected at highly homologous sites that had a 1 bp mismatch to the target site outside of the seed region in other rice studies (Zhang et al. 2014, Xu et al. 2015). Similarly, off-target editing was found at a frequency <3.0% between sites, with a 1 bp mismatch at the ninth nucleotide in the seed region in wheat (Zhang et al. 2016) and off-targets can also be identified in maize but can be decreased with the use of ribonucleoprotein (RNP) complexes (Svitashev et al. 2016). The take-home message is that the specificity of SpCas9 is species specific and needs to be separately evaluated, preferably with unbiased off-target assessment methods, but off-target events are unlikely to be as common in plants as with animals. Some gRNAs do not work at all. In our lab, we estimate that about 5% of gRNAs do not function for reasons unknown (Peterson et al. 2016, Čermák et al. 2017). Chromatin accessibility and nucleosome occupancy can decrease the ability of Cas9 to bind DNA in animal studies (Kuscu et al. 2014, Wu et al. 2014, Horlbeck et al. 2016). However, despite its effect on binding efficiency, chromatin accessibility does not negate Cas9 double-stranded DNA cleavage per se (Hsu et al. 2013, Moreno-Mateos et al. 2015). We have yet to find a gene that cannot be targeted by Cas9 and suspect that chromatin accessibility is unlikely to be a critical limiting factor in most plant targeting experiments. Delivery of gRNAs and Cas9 to Plant Cells Transgene-based delivery systems Also central to all editing applications is the need to get gRNAs and Cas nucleases into plant cell nuclei. Agrobacterium-mediated transformation of transgene-delivered CRISPR components is the most common method and varies across species, including floral dip in Arabidopsis, callus and immature embryo transformation, leaf tissue transformation and regeneration, and hairy root transformation. The difference between Agrobacterium transformation methods can have a profound effect on downstream editing success, especially in gene knockout experiments. To summarize broadly, plants that use callus-based regeneration for transformation frequently yield transgenic plants homozygous for indels in target genes in the first generation with Cas9, while plants such as Arabidopsis, which use floral dip transformation methods, typically do not (discussed below). Recently a magnetic-based approach to pollen transformation, using transgenes loaded onto magnetic particles which are driven into pollen grains via magnetic field exposure, has been used to transform pollen in cotton and other species (Zhao et al. 2017). In theory this approach could be used to deliver CRISPR components and should be applicable to a broad range of species. Individual transgenic gRNA expression requires RNA polymerase III promoters, with U3 and U6 promoters being the most commonly used. gRNA transcription starts at an adenine or guanine nucleotide, respectively, for these promoters, but genomic target sites need not start with a guanine or adenine on the 5' end to be targeted, so long as the nucleotide is included in the promoter::gRNA construct (Shan et al. 2013, Ma et al. 2015). Generally, editing efficiency can be increased by using a U6 promoter native to the species being edited (Sun et al. 2015). However, in cases where the U6 promoter is similar enough, as with tomato and Arabidopsis, using the Arabidopsis U6 promoter may be sufficient (Pan et al. 2016). For multiplexing, gRNAs can be expressed in linear arrays of Pol III promoter::gRNA ‘units’ in a single transgene in a head to tail orientation, with 16 gRNA units being the most we have delivered to Arabidopsis in a single transgene to date (Z.L.N., unpublished). Alternatively, multiple gRNAs can be encoded in a single polycistronic mRNA which is then processed to individual units by cis-elements that allow processing and release of individual gRNAs. One advantage is that polycistronic RNAs allow the use of POL II promoters for transcription, permitting cell-specific expression in theory. Polycistronic systems include self-cleaving ribozymes (Gao and Zhao 2014), and also systems that depend on accessory enzymes for gRNA release. For example, the Csy4 endoribonuclease expressed with Cas9 can be used to process gRNAs that are cleavable by Csy4 (Tsai et al. 2014). Using the endogenous tRNA processing system, multiplex genome editing was successfully achieved in rice with efficiencies up to 100% (Xie et al. 2015). There are additional species-specific effects on gRNA expression. For example, expression of gRNAs was lower in Arabidopsis compared with rice, possibly as a result of gRNA silencing (Jiang et al. 2014, Ma et al. 2015). In mammals, higher gRNA expression levels have been correlated with increased off-target editing. If gRNA expression is too high, reduction can be accomplished by inclusion of a UUU stretch in the seed region, which may lead to partial transcriptional termination and lower expression levels (Wu et al. 2014). Given the higher degree of specificity in plants, it is unlikely that gRNA expression levels are a general concern. In order to facilitate DNA cleavage, all systems to date employ Cas9 containing nuclear targeting sequences. To drive expression of Cas9, many systems use a constitutive promoter, such as Cauliflower mosaic virus (CaMV) 35S, or monocot- or dicot-specific versions of Ubiquitin promoters (Feng et al. 2013, Jiang et al. 2013, Miao et al. 2013, Shan et al. 2013, . Wang et al. 2014, Peterson et al. 2016). For floral dip transformation methods in Arabidopsis, it is not recommended to use the 35S promoter as this will prevent Cas9 expression in meristematic and germline cells of the shoot, precluding heritable genetic changes from being passed to progeny. Promoters that are active in callus are critical for callus regeneration methods and may be tied to the highly efficient generation of editing events in the T1 generation in callus-regenerated species (see below). Expression of Cas9 from egg cell-specific promoters may be used to restrict editing to the egg cells and one-cell stage embryos (Wang et al. 2015). Additional germline-specific promoters for Cas9 have been tested in Arabidopsis, including the SPL promoter, which targets sporogenous cells and microsporocytes, the LAT52 promoter, which targets pollen cells, and the DDT45 promoter, which targets egg cells (Mao et al. 2016). DDT45 was capable of producing heritable mutations in the T1 generation (Mao et al. 2016). Meiosis-specific promoter-driven expression of Cas9 has also been used to restrict editing (Eid et al. 2016). It is critical to keep in mind that the choice of promoter has strong effects on the ability to recover and select mutants, particularly in gene knockout experiments. The different expression systems used in CRISPR/Cas9 editing of plant genomes are covered in more detail by Lowder et al., who provide an excellent summary of the topic (Lowder et al. 2016). Several groups have published finished or assembly-ready cloning systems (Lowder et al. 2015, Ma et al. 2015, Čermák et al. 2017). Provided any particular toolkit uses appropriate promoters and selection, and have proven to work in planta and recover heritable mutants, user preference should govern choice. However, when it comes to multiplexing with transgene delivery, the need to create linear arrays of gRNAs, either with their own promoter or from a single cleavable transcript, poses cloning challenges. Golden Gate and Gibson assembly methods offer significant advantages in flexibility and cost (Lowder et al. 2015, Ma et al. 2015) and have been able to assemble as many as 12 gRNAs into a single vector (Čermák et al. 2017). Gene synthesis of gRNA arrays, followed by blunt-end cloning assembly, have allowed for the creation of 14 gRNA arrays in a single transgene, but is more expensive and slower than Golden Gate assembly (Peterson et al. 2016). Non-transgene delivery systems Biolistic delivery of Cas9/gRNA constructs has been successfully used in rice (Shan et al. 2013). Biolistic delivery methods can have higher rates of incomplete incorporation of the Cas9 transgene upon transformation when compared with Agrobacterium-mediated transformation, as seen in soybean (Jacobs et al. 2015). However, biolistic delivery methods are superior to Agrobacterium-mediated transformation when the goal is gene insertion via HDR, possibly a reflection of the number of DNA molecules delivered (Svitashev et al. 2015). More recently, the same group demonstrated that a pre-assembled Cas9 protein–gRNA RNP complex can be successfully introduced into maize embryos via particle bombardment with higher specificity compared with a non-RNP treatment (Svitashev et al. 2016). RNPs have been used in protoplasts to regenerate edited lettuce (Woo et al. 2015). While originally limited to plants that could be regenerated via protoplasts, methods for RNP delivery to immature embryos are now established in wheat and maize (Svitashev et al. 2016, Liang et al. 2017). Alternatively, if a species is recalcitrant to protoplast regeneration, either transiently expressed CRISPR/Cas9 DNA (TECCDNA) or transiently expressed CRISPR/Cas9 in vitro transcripts (TECCRNAs) can be delivered to immature embryos. In wheat, this system has been successfully used to generate homozygous mutations in the T0 generation (Zhang et al. 2016). Compared with RNP delivery into immature embryos, TECCDNA is more efficient at inducing on-target mutations, although RNPs yield a better on-target to off-target editing ratio (Liang et al. 2017). Cas Nuclease Options SpCas9 has been extensively studied with the goal of improving function in animal systems. There are many available plant codon-optimized versions of SpCas9, some which are broadly optimized for plants in general (Li et al. 2013, Xu et al. 2014, Ma et al. 2015), others that are optimized specifically for monocots (Jiang et al. 2013) and still others that are specifically optimized for a particular species, such as rice, tobacco or Arabidopsis (Jiang et al. 2013, Miao et al. 2013, Shan et al. 2013, Fauser et al. 2014, Gao et al. 2015, Peterson et al. 2016), and may lead to higher expression levels and higher editing efficiencies in some cases (Li et al. 2013, Xu et al. 2014). As mentioned above, in some plants, wild-type SpCas9 is exquisitely specific to on-target editing and, even in plants where off-target events are seen, the rates are very low. However, protein engineering has been used to improve SpCas9 specificity to reduce off-target editing while maintaining on-target efficiency in human cells. The most specific of these newly designed variants are high-fidelity (SpCas9-HF1) (Kleinstiver et al. 2016), enhanced specificity [eSpCas9(1.1)] (Slaymaker et al. 2016) and hyper accurate Cas9 (HypaCas9) (J.S. Chen et al. 2017). Comprehensive comparisons of these variants have not been done in plants, but could help reduce off-target rates further in plant species with higher off-target rates. SpCas9 proteins with mutations in the RuvC or HNH catalytic sites result in nickase mutants that exclusively cut either the plus or minus DNA strand (Gasiunas et al. 2012). The use of a single nickase is suitable for inducing HDR, but not for inducing NHEJ, as demonstrated in Arabidopsis (Fauser et al. 2014). However, if two Cas9 nickases are directed by a pair of gRNAs targeting opposite strands of a genomic target, it is possible to generate DSBs to be repaired via NHEJ (Ran et al. 2013). Since two gRNAs are required to identify and bind to appropriate targets, this method greatly decreases off-target editing events (Ran et al. 2013). One drawback is that the specific positioning of the paired targets decreases the number of usable target sites. Paired nickases have been used successfully to edit Arabidopsis and rice (Schiml et al. 2014, Mikami et al. 2016). Editing efficiency can be improved through the optimization of the distance between two gRNAs, and by using nickases that generate either 5' or 3' overhangs, depending on whether NHEJ or HDR is favored (Mikami et al. 2016). Similarly, catalytically inactive Cas9 can be paired with the nuclease domain of the FokI restriction enzyme to create a dimer that will nick DNA, which increases specificity due to the Fok1 dimerization requirements (Guilinger et al. 2014, Tsai et al. 2014). There are many Cas9 orthologs and alternative CRISPR effector nucleases. Some variants recognize different PAM sequences, cut DNA in a different manner or even target RNA. However, these may come with different efficiencies and specificities, or are not as well characterized as SpCas9. For most experiments, NGG PAM sites are readily found in target genes and wild-type SpCas9 will suffice. For very specific targeting of small genes or DNA elements, having different PAM options can be advantageous. SpCas9 preferentially recognizes the PAM 5'-NGG-3' but it will also recognize NAG, NGA, NAA, NGT, NGC and NCG at much lower frequencies, which is probably not practical (Hsu et al. 2013, Tsai et al. 2015). However, engineered versions of SpCas9 exist that preferentially recognize alternative PAM sites, including NGCG, NGAN and NGCG (Kleinstiver et al. 2015). Orthologs, such as SaCas9 from Streptococcus aureus, recognize the PAM sequence NNGRRT (Ran et al. 2015), and can induce editing in Arabidopsis (Zhang et al. 2017). Cas9 varieties found in Streptococcus thermophilus and Brevibacillus laterosporus have also been used successfully in Arabidopsis and maize, respectively (Karvelis et al. 2015, Steinert et al. 2015). In addition to recognizing alternative PAMs, other versions of Cas9 are in some cases smaller in size than SpCas9, which may help in some delivery systems (Steinert et al. 2015). Additionally, multiple Cas9 systems can be used simultaneously in order to maximize the number of sites targeted (Zhang et al. 2017). Having alternatives to NGG PAM sites expands options; however, it remains unclear if these variants will be as specific as wild-type SpCas9 in plants in many cases. Higher off-target editing has been observed in Arabidopsis when using SaCas9, though this may be an artifact of using gRNA design programs optimized for SpCas9 (Zhang et al. 2017). Further species-specific work is necessary to make broad conclusions in most cases. Cpf1 (CRISPR from Prevotella and Francisella1) is a single RNA-guided endonuclease that recognizes the PAM TTN and generates staggered DSBs, unlike SpCas9 (Zetsche et al. 2015). In rice, the Cpf1 system has successfully been used to generate heritable mutations with no off-target editing (Xu et al. 2017). Cpf1 is a useful alternative to SpCas9 because it generates larger deletions than SpCas9, as observed in soybean and tobacco protoplasts (Kim et al. 2017). It is important to note that when using Cas9, a trans-acting in vitro transcribed or chemically synthesized cRNA is necessary to generate mutations, while Cpf1 is guided by a single cRNA without the need for a transacting cRNA to induce mutations (Kim et al. 2017, Xu et al. 2017). Cas13a derived from Leptotrichia wadei is an RNA-guided RNase shown to be effective in reducing target transcript accumulation by >50% in rice protoplasts (Abudayyeh et al. 2017). This exciting finding opens up the possibility of robust RNA-mediated RNA targeting in plants, although it remains to be seen how effective Cas13a will be in producing phenotypic knockdowns in whole plants. A General Comment on Efficiency In general, the editing efficiency and specificity of a particular approach must be evaluated at the level of stable plant generation in the species of interest. Extrapolation of rules derived from protoplasts and other species does not necessarily insure similar success levels across platforms. Most importantly, there is considerable variability in gRNA efficiency, which does not appear to change with expression system or Cas9 delivery method and is often hard to predict. Despite all the modifications and alternatives, for many applications SpCas9 will suffice and there may be no clear advantage for one system over another in efficiency or specificity. This is especially true in single gene mutation targeting in callus-regenerating plants, which typically yields high rates of homozygous mutant recovery, or in Arabidopsis when considering specificity. In those cases, the ease of use of the system and its record of validation in planta might be the strongest consideration. Recent work provides evidence that heat shock treatment of plants at 37°C for brief periods of time can enhance rates of Cas9 editing (LeBlanc et al. 2018). This effect is apparent in both somatic and germline tissues, where as much as 100-fold increases of editing rates are seen. This effect is linked to higher efficiency of Cas9 double-stranded DNA cleavage at 37°C. Excitingly, this simple treatment works in multiple species, suggesting that this may be an excellent tool to bolster editing experiments across systems. Considerations for Creating Loss-of-Function Gene Mutations One of the simplest uses of CRISPR in plants has been to generate heritable knockout mutants to assess gene function, but this approach has several considerations. One of the main considerations is whether to delete a gene entirely or create a frameshift mutant using a smaller indel. The majority of Cas9 indels occur at the –3 site to the PAM sequence in the protospacer sequence, where Cas9 cuts. In tomato, some mutations were reported to occur between the fourth and fifth base upstream of the PAM, 1 bp further than typically expected (Doudna and Charpentier 2014, Pan et al. 2016). A few cases of nucleotide substitutions have been noted in rice and Arabidopsis arising from repair (Feng et al. 2014, Zhang et al. 2014, Ishizaki 2016). For frameshift-induced indels, targeting the first exon closest to the initiator methionine can usually suffice to truncate function early. For instance, targeting the CORYNE gene with a single gRNA in Arabidopsis resulted in a truncation at amino acid 6 out of 402, a clear null (Nimchuk 2017). In some cases, collateral nonsense-mediated mRNA decay may accompany frameshift mutants (Copenhaver, personal communication). If two distinct gRNAs are used to target exons simultaneously, it can insure editing should one gRNA not work, and provide a broader collection of mutant alleles. However, if the Cas9 transgene is still functioning in a single editing site mutant, phenotypic revertants can arise when deletions and/or insertions at the second editing site restore the frame in rare cases (Z.L.N. unpublished). Typically indels are small, mostly single base pair changes across species (Feng et al. 2014, Zhang et al. 2014, Ma et al. 2015, Svitashev et al. 2015, Ishizaki 2016, Pan et al. 2016) (Fig. 2A). Larger deletions occur at increasingly lower frequencies and therefore to create a true complete deletion of a gene, dual gRNAs flanking the gene are typically required. Such large-scale deletions, or two-site deletions, can be generated when editing occurs at two nearby target sites simultaneously and endogenous repair mechanisms repair the DNA breaks by ligating the 3' end of one target with the 5' end of the other (Moreno-Mateos et al. 2015) (Fig. 2C). Two-site deletions have been successfully induced in rice, Arabidopsis, tobacco and tomato (Brooks et al. 2014, Feng et al. 2014, Zhou et al. 2014, Lowder et al. 2015, Ma et al. 2015, Xie et al. 2015, Gao et al. 2016, Mikami et al. 2016). Gene deletion frequencies differ strongly across protoplasts, callus and floral dip regeneration. Callus regeneration often gives rise to longer deletion events that occur between two gRNAs (Gao et al. 2016, Čermák et al. 2017, Rodríguez-Leal et al. 2017) while comparable deletions are recovered in T2 plants of Arabidopsis through floral dip at a low frequency; around 1% of plants in our hands (Z.L.N., personal observation) (Peterson et al. 2016). As such, PCR-based screening systems to enrich such plants will need to be performed, unless the phenotype is known and can be used to select mutant plants from the population. Fig. 2 View largeDownload slide CRISPR/Cas9 methods to alter gene function. The cartoon depicts various scenarios when using different Cas9 applications. (A) A double-stranded break at the target sequence activates the NHEJ pathway that results in the insertion or deletion of a random base at the site of repair, which causes a frameshift mutation. (B) Homology-directed repair mechanisms use homologous regions to rejoin cleaved DNA often with the introduction of donor DNA to create an intended modified DNA. (C) Cas9-induced large deletions are achieved by using multiple gRNAs flanking the gene of interest. Such large-scale deletions, or two-site deletions, can be generated when editing occurs at two nearby target sites simultaneously and endogenous repair mechanisms repair the DNA breaks by ligating the 3' end of one target with the 5' end of the other. (D) dCas9 acts as an artificial transcription factor when fused with a transcriptional activator or repressor. (E) A base editor, consisting of dCas9 and a cytidine deaminase converts C to U in a targeted base pair. The DNA repair pathway converts the resulting U:G mismatch to T:A. Fig. 2 View largeDownload slide CRISPR/Cas9 methods to alter gene function. The cartoon depicts various scenarios when using different Cas9 applications. (A) A double-stranded break at the target sequence activates the NHEJ pathway that results in the insertion or deletion of a random base at the site of repair, which causes a frameshift mutation. (B) Homology-directed repair mechanisms use homologous regions to rejoin cleaved DNA often with the introduction of donor DNA to create an intended modified DNA. (C) Cas9-induced large deletions are achieved by using multiple gRNAs flanking the gene of interest. Such large-scale deletions, or two-site deletions, can be generated when editing occurs at two nearby target sites simultaneously and endogenous repair mechanisms repair the DNA breaks by ligating the 3' end of one target with the 5' end of the other. (D) dCas9 acts as an artificial transcription factor when fused with a transcriptional activator or repressor. (E) A base editor, consisting of dCas9 and a cytidine deaminase converts C to U in a targeted base pair. The DNA repair pathway converts the resulting U:G mismatch to T:A. Plant Phenotypes with CRISPR/Cas9: Advantages and Challenges When using CRISPR/Cas9, different copies of a targeted gene may be mutated at different times. Depending on the Cas9 delivery system, this can occur at different stages and provides opportunities and challenges for isolating plants. When both copies of the targeted gene are mutated in an embryogenic cell before the initial division, the genotype may be homozygous, if the mutations are of the same nature, or biallelic, if two different mutations occurred in the two gene copies (Zhang et al. 2014). In callus-regenerated plant species, including tomato (Brooks et al. 2014, Pan et al. 2016), rice (Xu et al. 2015, Ishizaki 2016), lettuce (Woo et al. 2015) and wheat (Zhang et al. 2016), it is easy to detect homozygous biallelic mutations in T0 plants and with a high editing efficiency. Arabidopsis T1 mutants display an extremely low frequency of homozygous mutations in the first generation, even with germline-specific Cas9 promoters, with homozygous mutants recovering an increasing frequency in later generations (Fauser et al. 2014, Feng et al. 2014). In many cases, this provides a clear advantage for callus regeneration, except if the gene targeted is lethal, sterile or severely developmentally compromised when mutant. In these cases, recovery of heterozygous events may be necessary. Once editing has occurred or is inherited stably in germline cells, gRNA target sites will not re-cleave and are fixed. However, in plants not stably mutant, and still carrying Cas9-gRNA transgenes, considerable sectoring and chimerism in somatic cells is expected if Cas9 is expressed from a constitutive promoter (Fauser et al. 2014, Feng et al. 2014, Peterson et al. 2016). This occurs in first-generation transgenic plants, but also in subsequent generations (Fauser et al. 2014, Feng et al. 2014). Chimerism and mosaicism, without fixed heritable mutations, by themselves can give rise to phenotypes in plants when using constitutive Cas9 expression and to unpredictable phenotype ratios, often varying between transgenic lines (Zhu et al. 2014, Ishizaki 2016, Pan et al. 2016). Mosaic-induced phenotypes can arise in some cases at a very high rate even in T1 plants (Fig. 3). As such, phenotype alone does not insure plants are stable homozygous mutants. Fixed, homozygous mutants must be confirmed by individual plant sequencing and confirming Cas9 absence by segregating away Cas9 transgenes. In some cases, mosaicism can be advantageous, allowing a rapid estimate of what relevant phenotypes might be, or result in plants that are intermediate with null mutants and may help assign gene function. In other cases, sectoring can occur that may help guide homozygous mutant recovery. For instance, targeting of CORYNE in Arabidopsis gave rise to a single branch with all mutant flowers that arose from a stably mutated biallelic sector that propagated clonally. Collection of seed from that branch yielded all fixed mutants in the next generation, many Cas9 free, and two different alleles (Nimchuk 2017). In other cases, as noted above, strong mosaicism may be phenotypically deleterious, and germline editing can be used to identify heterozygous mutants in the first generation using promoters restricted to the germline to drive Cas9. Mosaicism can also be avoided by using pre-assembled CRISPR/Cas9 RNPs since they cleave chromosomal target sites immediately upon transfection and are presumably degraded by endogenous proteases within the cells over time (Woo et al. 2015). Fig. 3 View largeDownload slide Ubiquitous Cas9/gRNA expression can yield mosaic target gene editing and intermediate phenotypes. Null mutations in the WUSCHEL (WUS) gene in Arabidopsis severely compromise shoot growth and strongly reduce flower formation, and prevent fruit development (right). In contrast, mosaic editing of WUS with ubiquitously expressed Cas9 and WUS-targeting gRNA results in intermediate phenotypes displaying robust shoot growth, flower formation, but with missing floral organs at high frequencies (60% of transgenic plants). Left: close up of wild-type and Cas9/WUS gRNA-targeted inflorescences to show detail. Yellow arrows, mature fruit derived from individual flowers in the wild type; blue arrow, flower lacking fruit formation. Note the lack of flower development on wus null mutants. Fig. 3 View largeDownload slide Ubiquitous Cas9/gRNA expression can yield mosaic target gene editing and intermediate phenotypes. Null mutations in the WUSCHEL (WUS) gene in Arabidopsis severely compromise shoot growth and strongly reduce flower formation, and prevent fruit development (right). In contrast, mosaic editing of WUS with ubiquitously expressed Cas9 and WUS-targeting gRNA results in intermediate phenotypes displaying robust shoot growth, flower formation, but with missing floral organs at high frequencies (60% of transgenic plants). Left: close up of wild-type and Cas9/WUS gRNA-targeted inflorescences to show detail. Yellow arrows, mature fruit derived from individual flowers in the wild type; blue arrow, flower lacking fruit formation. Note the lack of flower development on wus null mutants. Ultimately it is necessary to sequence genes, and confirm phenotypes in plants lacking Cas9 transgenes by genetic segregation, before concluding that phenotypes are associated with heritable mutations. PCR and/or drug selection to determine T-DNA presence in T2 generations is a relatively straightforward approach. Seed-expressed mCherry cassettes on Cas9 transgenes can allow for the rapid identification of Cas9-free plants in the T2 generation (Gao et al. 2016). Screening for mutations can be done by direct sequencing, and this might be the easiest method if an obvious or expected phenotype arises in T2 plants in Arabidopsis, and is standard in T0 callus regeneration systems where homozygous mutant recovery is high and plant numbers are low. However, to detect segregating T2 events when phenotypes are unknown, or in higher order multiplex events, or rare T1 events in Arabidopsis, a mutational screening strategy might be desirable. When multiplexing in Arabidopsis, the ability to recover higher order T2 mutants depends on the gene editing efficiency of each of the gRNAs. As such, recovery of higher order mutants and inheritance patterns can be highly variable, thus a good screening strategy is advisable. gRNAs that potentially disrupt an endogenous restriction enzyme site can be designed and mutants can be identified using a restriction fragment length polymorphism assay (Nekrasov et al. 2013, Shan et al. 2013, Kim et al. 2014). However, this approach limits gRNA site selection options. Diagnostic restriction sites can be created using primers that incorporate nucleotide changes, similar to the derived cleaved amplified polymorphic sequences (dCAPS) (Neff et al. 1998) used with missense mutations, and mutants can be identified on this basis (Endo et al. 2015, Nimchuk 2017). This approach has been recently automated by the design of web-based programs that can identify primers that create diagnostic restrictions sites (indCAPS) (Hodgens et al. 2017). Critically, this program tolerates indels, unlike standard dCAPS identification programs. The T7 endonuclease I assay and Surveyor system can be used to identify mutations based on heteroduplex mismatches between wild-type and mutant DNA sequences (Qiu et al. 2004, Kim et al. 2009, Xie and Yang 2013, Y. Wang et al. 2014); however, these assays are more costly and require additional PCR and reaction steps, and are not able to distinguish between wild-type and biallelic homozygous mutations or biallelic and monoallelic heterozygous mutants (Kim et al. 2014). Heteroduplex DNA formation can also be used in melting curve analysis to identify indel events (Denbow et al. 2017). Since homoduplex DNA migrates at a faster rate than heteroduplex DNA due to the open angle located between the matched and mismatched heteroduplex strands caused by mutation, this feature can also be used to detect editing visually, using polyacrylamide gel electrophoresis (PAGE) (Zhu et al. 2014). In rice, the PAGE assay has been modified to use single-stranded DNA since it offers the advantages of higher precision and the ability to distinguish homozygous mutants from the wild type (Zheng et al. 2016). Alternatively, if larger deletions are expected, then PCR products can be run on an appropriate percentage agarose gel, or even polyacrylamide gels. However, these results can be easily misinterpreted because small indels can yield wild-type-appearing bands and, occasionally, the slower migrating heteroduplex products will mistakenly appear as insertions (Brooks et al. 2014). In our hands, indCAPS and direct sequencing are the most robust and cost-effective way to screen for mutations, although plate-based Sanger sequencing of T2 plants can also provide a rapid and complete approach if time is more of an issue and cost is not. Multiple, independently derived alleles usually insure that phenotypes are associated with specific gene mutations, and complementation experiments should be performed if necessary. However, for extreme higher order multiplex mutations, which can be upwards of 10 genes in some cases and take several generations, technical and financial restrictions make complete independent mutation set identification and comprehensive complementation experiments challenging. Many genes important for crop traits are derived from mutations that alter but do not eliminate gene function, often resulting in cis regulatory mutations. A different approach for altering gene function via CRISPR is the targeting of wild-type Cas9 to endogenous promoter elements, rather than the coding region of the gene of interest itself, to create indels in promoter sequences that alter gene expression and function. Specific promoter elements, if known, can be targeted, but promoter sequences can also be randomly mutated in a blind fashion by exploiting the ability to multiplex with Cas9. This powerful random approach was recently demonstrated in tomato using suites of gRNAs spaced across different promoter sequences. This resulted in populations of plants with a continuum of non-null phenotypes arising from a variety of promoter deletion/insertion events between gRNAs which often altered target gene expression levels. This exciting promoter targeting approach is likely to be highly advantageous for crop breeding as it can rapidly create a broad range of trait variants that can be exploited for breeding (Rodríguez-Leal et al. 2017). Repairing and Replacing Genes with CRISPR in Plants The generation of mutations by CRISPR is dependent on the ability of the host species to repair DNA breaks. The most frequently observed repair mechanism is NHEJ, which generates unpredictable indels at specific target sites. A less frequently used repair mechanism is HDR, which can replace a stretch of sequence with an externally supplied sequence of choice, also known as gene repair. Repair via HDR is advantageous for creating knock-in mutants, which include applications such as substitution of a promoter for increased expression of a stress response gene (Shi et al. 2017) and the modification of herbicide resistance genes (Svitashev et al. 2015, Endo et al. 2016, Sun et al. 2016). While CRISPR editing in plants has predominantly focused on NHEJ events, multiple species have been successfully edited via HDR, including tomato (Čermák et al. 2015), rice (Endo et al. 2016, Sun et al. 2016, Wang et al. 2017), wheat (Gil-Humanes et al. 2017), maize (Svitashev et al. 2015, Shi et al. 2017), Arabidopsis (Zhao et al. 2016) and soybean (Li et al. 2015), among others. Generally, the editing efficiency of gene targeting is significantly lower for indel generation, with efficiencies typically <1% (Svitashev et al. 2015, Endo et al. 2016). This may suffice if a clear phenotype or selection for the replacement is available, but in most cases these rates would necessitate considerable screening of plants. NHEJ repair pathways compete with HDR and, in plants, there appears to be a strong bias towards NHEJ, complicating gene replacement methods (Qi et al. 2013). Additional barriers include the temporal delivery of the components to dividing cells, where the HDR pathway predominantly functions, and the complexity of the components needed for gene repair and replacement. Repairing or replacing genes through HDR requires Cas9, CRISPR target site(s) flanking, or within, the desired insertion site, and a DNA sequence, often called the ‘donor DNA’, to be inserted (Fig. 2B). The sequence to be inserted is typically flanked on the 5' and 3' ends, with homology arms, or sequences that are homologous to the native genomic region, upstream and downstream of the insertion site. Homology arms from 46 bp to 1 kb in length have been used successfully (Li et al. 2015, Svitashev et al. 2015, Sun et al. 2016, Gil-Humanes et al. 2017, Shi et al. 2017, Wang et al. 2017). While these additional DNA sequences are required for gene replacement, they further complicate the transformation process, as the efficiency of Agrobacterium-mediated transformation drops with an increase in the size of the vector or may lead to unwanted insertion of vector DNA. Methods circumventing these particular challenges include: transformation through particle bombardment (Li et al. 2015, Svitashev et al. 2015, Sun et al. 2016, Shi et al. 2017), sequential Agrobacterium transformation (Endo et al. 2016) and Agrobacterium-mediated transformation with a geminiviral vector (Čermák et al. 2015, Gil-Humanes et al. 2017, Wang et al. 2017).The use of geminiviral vectors increases editing efficiency, making this technique more feasible (Gil-Humanes et al. 2017, Wang et al. 2017). There is considerable interest in increasing the efficiency of gene repair and replacement, and strategies promoting HDR have been explored extensively in animals. These strategies include reducing the effectiveness of NHEJ by genetic or pharmacological targeting of NHEJ pathway components, using Cas effectors or combinations of nickases to avoid creation of a DSB, and targeting dividing cell populations (Pawelczak et al. 2017). Genetically manipulating NHEJ in Arabidopsis has increased gene efficiencies when using zinc finger nucleases. These mutants have not been used in conjunction with CRISPR/Cas9, but the technique shows promise for increasing the efficiency of gene repair and replacement in plants (Li et al. 2007, Qi et al. 2013, Zhang et al. 2017). Sigma-Aldrich maintains a nice collection of information on drugs used to bias HDR in CRISPR applications, but to our knowledge most of these have not been characterized in plants. Homing Applications of Cas Nucleases in Plants The RNA-based DNA homing ability of Cas9 independent of Cas9 nuclease activity (dead Cas9, dCas9) can be used to target dCas9 fusion proteins to DNA in a sequence-specific manner for various applications (Didovyk et al. 2016). Efficient targeted gene activation and repression have been achieved by the fusion of activation or repressor domains, respectively, to dCas9 (Fig. 2D) (Lowder et al. 2015, Piatek et al. 2015, Vazquez-Vilar et al. 2016). For single gene overexpression, it may be easier and more efficient to drive gene expression from heterologous promoters, but this approach could be particularly advantageous for multiplexed gene expression regulation, especially for functionally related genes in biochemical pathways. For single gene overexpression, it may be easiest to drive gene expression from heterologous promoters instead. The fusion of dCas9 to VP64 or VPR transcriptional activator domains increases transcriptional activation of endogenous targets (Chavez et al. 2015, Čermák et al. 2017) (Fig. 2C). The CRISPR–Cpf1 system has also been used for targeted gene repression in Arabidopsis with transcriptional reductions of up to 10-fold (Tang et al. 2017). In addition to creating DSBs, Cas9 can also be exploited to mutate nucleotides, or base edit. In base editing experiments, dCas9 fusions are made with cytosine deaminase domains allowing gRNA-mediated targeting of the fusion protein to genomic DNA and the creation of nucleotide substitutions rather than indels (Hess et al. 2017) (Fig. 2E). At the targeted DNA sites, cytosine deaminase acts upon cytosines near or within the targeted DNA, resulting in heritable conversions of C–G pairs to A–T pairs. The base editing approach has been used in several plant species and could be useful for creating allelic series in target genes or for protein structure–function analysis (Y. Chen et al. 2017, Shimatani et al. 2017, Zong et al. 2017). In animals, there is considerable interest in improving the rates of base editing and determining which cytosines in a targeted DNA region will be modified, as base editors tend to act on cytosines near or in targeted regions in an indiscriminant manner. Exciting recent work has resulted in the creation of adenine base editors, allowing A–T to G–C conversions (Gaudelli et al. 2017). The use of these editors in plants remains to be explored but opens up several new avenues for genome editing. Discussion The CRISPR/Cas9 system is the most recent and exciting addition to the genome editing toolbox because of its simplicity and successful application. The wide array of CRISPR/Cas9 optimizations allows the system to be used for multiple purposes with increasing efficiency and specificity. Newer technologies enabling epigenetic and transcriptional regulation should complement more traditional methods and could be used to tune or multiplex plant gene expression with unprecedented control. The ability to base edit in plants is in its infancy but, considering the potential applications for targeted in vivo substitution mutagenesis, represents an exciting opportunity. The massive increase in CRISPR systems and tools has begun to change what we can do with model plants. There is likely to be a resurgence in defining plant gene function, particularly for very small genes, discrete DNA elements and highly redundant gene families arising from this technology. The technology has even changed what a model system is, and should revolutionize comparative plant evolution studies with its ability to define gene function in traditionally non-model plants. Lastly, the ability to precisely target and modify gene functions in crops should provide a key tool in the hunt for new traits to meet the challenges facing global food production. Given the broad interest and push in CRISPR systems, new approaches to increasing efficiency, specificity, heritability and new CRISPR variants and applications are inevitable in the near future. It is an exciting time to be involved in plant research. Funding This work was supported by the National Science Foundation [grant No. IOS-1546837 to Z.L.N.]. Acknowledgments We would like to extend an apology to colleagues whose research we were unable to discuss due to length limitations. We thank Drs. Joe Kieber, Carly Sjogren and Ashley Crook for comments on the manuscript. Disclosures The authors have no conflict of interest to declare. References Abudayyeh O.O. , Gootenberg J.S. , Essletzbichler P. , Han S. , Joung J. , Belanto J.J. , et al. ( 2017 ) RNA targeting with CRISPR–Cas13 . Nature 550 : 280 – 284 . Google Scholar CrossRef Search ADS PubMed Anders C. , Bargsten K. , Jinek M. ( 2016 ) Structural plasticity of PAM recognition by engineered variants of the RNA-guided endonuclease Cas9 . Mol. Cell 61 : 895 – 902 . Google Scholar CrossRef Search ADS PubMed Barrangou R. , Fremaux C. , Deveau H. , Richards M. , Boyaval P. , Moineau S. , et al. ( 2007 ) CRISPR provides acquired resistance against viruses in prokaryotes . 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Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations CAPS cleaved amplified polymorphic sequence Cpf1 CRISPR from Prevotella and Francisella CRISPR/Cas9 clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 crRNA CRISPR RNA DSB double-stranded break dsODN double-stranded oligodeoxynucleotide gRNA guide RNA HDR homology-directed repair NHEJ non-homologous end joining PAM protospacer adjacent motif RNP ribonucleoprotein SaCas9 Streptococcus aureus Cas9 SpCas9 Streptococcus pyogenes Cas9 TECCDNA transiently expressed CRISPR/Cas9 DNA TECCRNA transiently expressed CRISPR/Cas9 RNA tru-gRNA truncated guide RNA tracrRNA trans-activating CRISPR RNA © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Cutting Edge Genetics: CRISPR/Cas9 Editing of Plant Genomes

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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10.1093/pcp/pcy079
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Abstract

Abstract The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system is a genome editing technology transforming the field of plant biology by virtue of the system’s efficiency and specificity. The system has quickly evolved for many diverse applications including multiplex gene mutation, gene replacement and transcriptional control. As CRISPR/Cas9 is increasingly applied to plants, it is becoming clear that each component of the system can be modified to improve editing results. This review aims to highlight common considerations and options when conducting CRISPR/Cas9 experiments. Introduction The ability to manipulate gene function has been an indispensable tool for basic plant research and for the generation of improved crop varieties. Whether in the lab or in the field, the ability to modulate gene function would ideally employ technology that is specific, efficient and heritable. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) genome editing technology provides such a tool, enabling revolutionary advances in both arenas of plant biology. CRISPR/Cas systems are bacterial adaptable immune systems against foreign DNA sources such as bacteriophages or plasmids (Wiedenheft et al. 2012). CRISPR/Cas immunity is mediated by RNA-guided nucleases, which target invading phage genomes for degradation in a sequence-specific manner, conferring immunity. There are five distinct CRISPR/Cas systems identified in bacteria which differ in their gene composition and ability to cleave phage DNA, RNA or both. These systems have been co-opted for genome editing in heterologous species for their ability to confer sequence-specific targeting and cleavage of DNA, or RNA, targets. The Type II mechanism is the most commonly used in genome editing for heterologous systems because it requires only one Cas protein, Cas9, for DNA targeting and cleavage (Deltcheva et al. 2011). CRISPR/Cas immunity in bacteria consists of an adaptation phase, an expression (or biogenesis) phase and an interference phase (Jiang and Doudna 2016). During the adaptation phase, a DNA record of an attempted phage infection is created when an invading phage genome is cleaved by endonucleases into short DNA sequences, which are then stored in the bacterial genome in so-called CRISPR DNA arrays (Barrangou et al. 2007). The CRISPR DNA array consists of short conserved sequence repeats separated by short variable sequences of spacer DNA, each corresponding to a unique captured foreign DNA sequence. During the expression phase, the entire CRISPR array is transcribed and processed into small CRISPR RNAs (cRNAs), consisting of one captured spacer sequence and one repeat sequence each (Jiang and Doudna 2016). A cRNA hybridizes with a second RNA, the trans-activating CRISPR RNA (tracrRNA), to form a small hybrid RNA molecule, the crRNA (Deltcheva et al. 2011). During the interference phase, the mature crRNA hybrid binds the Cas9 endonuclease and is recruited to invading phage DNA (Jiang and Doudna 2016). Binding of the Cas nuclease–crRNA complex is dependent on two factors: (i) complementary base pairing between the spacer sequences on the crRNA and corresponding phage DNA ‘protospacer’ sequence; and (ii) a protospacer adjacent motif (PAM), a short nucleotide consensus sequence that flanks the protospacer sequence and is critical for Cas9 nuclease binding (Gasiunas et al. 2012, Jinek et al. 2012). Streptococcus pyogenes Cas9 (SpCas9) cleaves target DNA sites flanked by a PAM sequence conforming to a 5'-NGG-3' consensus immediately following the 3' end of the protospacer target, although alternative variant PAMs will be cleaved by Cas9 at lower efficiencies (Anders et al. 2016). The requirement for the PAM sequence in target binding by the Cas9–crRNA insures that the complex will not bind and cleave spacer sequences in the bacterial genomic CRISPR array, which lacks PAM sites. Pioneering work from the labs of Charpentier and Doudna showed that the ability to target and cleave DNA in a sequence-specific manner could be reduced in vitro down to just Cas9 and a single synthetic RNA which fuses the tracrRNA and crRNA together into a single guide RNA (gRNA) (Jinek et al. 2012). In this work, they demonstrated that from these simple principles gRNAs could be designed that target protospacers adjacent to PAM sites in a GFP (green fluorescent protein) gene, and Cas9 would then cleave the double-stranded GFP DNA. Subsequent work from the Church and Zhang groups extended these principles to human cells, showing that you could exploit these properties to target Cas9–gRNA complexes to DNA in a sequence-specific manner (Cong et al. 2013, MaLi et al. 2013). Eukaryotic gene function could be disrupted by creating double-stranded DNA breaks (DSBs) repaired incorrectly by non-homologous end-joining (NHEJ) DNA repair mechanisms, resulting in nucleotide insertions and/or deletions (indels) that generate frameshift mutations. In addition to donor DNA repair templates, Cas9-mediated DNA breaks could be exploited to knock-in donor DNA sequences by the homology-dependent repair (HDR) DNA repair pathway, effectively replacing endogenous gene sequences. Excitingly, distinct gRNAs targeting different genomic sites could be combined simultaneously to multiplex edit different genome sites at once. Together these three papers set off the current revolution in genome engineering, which quickly extended to plants. Central to any use of CRISPR systems in genome editing is the desire to edit target sites efficiently and specifically. As the use of Cas9 has expanded, different strategies have emerged that impact experimental success. Here we compare and discuss Cas9 approaches in plants, using current literature and experiences in our lab, and discuss caveats and considerations with this technology. A summary of the optimizable parameters of the CRISPR/Cas9 system for plants discussed in the present review can be viewed in Table 1. Table 1 Optimizable parameters in the design of CRISPR/Cas9 systems for plants Feature Optimization Options Reference(s) gRNA Design GC content Ma et al. 2015, Pan et al. 2016, Zhang et al. 2014 Target accessibility Kuscu et al. 2014, Wu et al. 2014 Number of mismatches Xing et al. 2014 Spacer length Osakabe et al. 2016 Delivery Systems Agrobacterium- mediated delivery Ma et al. 2015 Biolistic delivery Shan et al. 2013, Jacobs et al. 2015, Svitashev et al. 2015 RNP delivery Woo et al. 2015, Liang et al. 2017, Svitashev 2016 TECCDNA/TECCRNA delivery Zhang et al. 2016, Liang et al. 2017 Viral delivery Čermák et al. 2015, Gil-Humanes et al. 2017 Cas9 expression systems CaMV 35s promoter (for transient assays, not recommended for stable heritable events) Feng et al. 2013, Jiang et al. 2013, Shan et al. 2013 Species appropriate Ubiquitin promoter Miao et al. 2013, Wang et al. 2014, Peterson et al. 2016 Egg cell-specific (EC1.2) promoter Wang et al. 2015 SPL promoter Mao et al. 2016 LAT52 promoter Mao et al. 2016 DDT45 promoter Mao et al. 2016 Meiosis-specific (MGE1/MGE2/MGE3) promoter Eid et al. 2016 Cas9 modifications Codon optimization Fauser et al. 2014, Gao et al. 2015a, Jiang et al. 2013b, Miao et al. 2013, Shan et al. 2013 Paired nickases Fauser et al. 2014, Mikami et al. 2016, Schiml et al. 2014 dCas9 Lowder et al. 2015, Piatek et al. 2015 Heat shock treatment LeBlanc et al. 2018 Alternative endonuclease varieties SaCas9 Zhang et al. 2017, Steinert et al. 2015 St1Cas9 Steinert et al. 2015 SpCas9-HF1 Kleinstiver et al. 2016 eSpCas9(1.1) Slaymaker et al. 2016 HypaCas9 J.S. Chen et al. 2017 Cpf1 Xu et al. 2017, Kim et al. 2017 Cas13a Abudayyeh et al. 2017 Feature Optimization Options Reference(s) gRNA Design GC content Ma et al. 2015, Pan et al. 2016, Zhang et al. 2014 Target accessibility Kuscu et al. 2014, Wu et al. 2014 Number of mismatches Xing et al. 2014 Spacer length Osakabe et al. 2016 Delivery Systems Agrobacterium- mediated delivery Ma et al. 2015 Biolistic delivery Shan et al. 2013, Jacobs et al. 2015, Svitashev et al. 2015 RNP delivery Woo et al. 2015, Liang et al. 2017, Svitashev 2016 TECCDNA/TECCRNA delivery Zhang et al. 2016, Liang et al. 2017 Viral delivery Čermák et al. 2015, Gil-Humanes et al. 2017 Cas9 expression systems CaMV 35s promoter (for transient assays, not recommended for stable heritable events) Feng et al. 2013, Jiang et al. 2013, Shan et al. 2013 Species appropriate Ubiquitin promoter Miao et al. 2013, Wang et al. 2014, Peterson et al. 2016 Egg cell-specific (EC1.2) promoter Wang et al. 2015 SPL promoter Mao et al. 2016 LAT52 promoter Mao et al. 2016 DDT45 promoter Mao et al. 2016 Meiosis-specific (MGE1/MGE2/MGE3) promoter Eid et al. 2016 Cas9 modifications Codon optimization Fauser et al. 2014, Gao et al. 2015a, Jiang et al. 2013b, Miao et al. 2013, Shan et al. 2013 Paired nickases Fauser et al. 2014, Mikami et al. 2016, Schiml et al. 2014 dCas9 Lowder et al. 2015, Piatek et al. 2015 Heat shock treatment LeBlanc et al. 2018 Alternative endonuclease varieties SaCas9 Zhang et al. 2017, Steinert et al. 2015 St1Cas9 Steinert et al. 2015 SpCas9-HF1 Kleinstiver et al. 2016 eSpCas9(1.1) Slaymaker et al. 2016 HypaCas9 J.S. Chen et al. 2017 Cpf1 Xu et al. 2017, Kim et al. 2017 Cas13a Abudayyeh et al. 2017 Table 1 Optimizable parameters in the design of CRISPR/Cas9 systems for plants Feature Optimization Options Reference(s) gRNA Design GC content Ma et al. 2015, Pan et al. 2016, Zhang et al. 2014 Target accessibility Kuscu et al. 2014, Wu et al. 2014 Number of mismatches Xing et al. 2014 Spacer length Osakabe et al. 2016 Delivery Systems Agrobacterium- mediated delivery Ma et al. 2015 Biolistic delivery Shan et al. 2013, Jacobs et al. 2015, Svitashev et al. 2015 RNP delivery Woo et al. 2015, Liang et al. 2017, Svitashev 2016 TECCDNA/TECCRNA delivery Zhang et al. 2016, Liang et al. 2017 Viral delivery Čermák et al. 2015, Gil-Humanes et al. 2017 Cas9 expression systems CaMV 35s promoter (for transient assays, not recommended for stable heritable events) Feng et al. 2013, Jiang et al. 2013, Shan et al. 2013 Species appropriate Ubiquitin promoter Miao et al. 2013, Wang et al. 2014, Peterson et al. 2016 Egg cell-specific (EC1.2) promoter Wang et al. 2015 SPL promoter Mao et al. 2016 LAT52 promoter Mao et al. 2016 DDT45 promoter Mao et al. 2016 Meiosis-specific (MGE1/MGE2/MGE3) promoter Eid et al. 2016 Cas9 modifications Codon optimization Fauser et al. 2014, Gao et al. 2015a, Jiang et al. 2013b, Miao et al. 2013, Shan et al. 2013 Paired nickases Fauser et al. 2014, Mikami et al. 2016, Schiml et al. 2014 dCas9 Lowder et al. 2015, Piatek et al. 2015 Heat shock treatment LeBlanc et al. 2018 Alternative endonuclease varieties SaCas9 Zhang et al. 2017, Steinert et al. 2015 St1Cas9 Steinert et al. 2015 SpCas9-HF1 Kleinstiver et al. 2016 eSpCas9(1.1) Slaymaker et al. 2016 HypaCas9 J.S. Chen et al. 2017 Cpf1 Xu et al. 2017, Kim et al. 2017 Cas13a Abudayyeh et al. 2017 Feature Optimization Options Reference(s) gRNA Design GC content Ma et al. 2015, Pan et al. 2016, Zhang et al. 2014 Target accessibility Kuscu et al. 2014, Wu et al. 2014 Number of mismatches Xing et al. 2014 Spacer length Osakabe et al. 2016 Delivery Systems Agrobacterium- mediated delivery Ma et al. 2015 Biolistic delivery Shan et al. 2013, Jacobs et al. 2015, Svitashev et al. 2015 RNP delivery Woo et al. 2015, Liang et al. 2017, Svitashev 2016 TECCDNA/TECCRNA delivery Zhang et al. 2016, Liang et al. 2017 Viral delivery Čermák et al. 2015, Gil-Humanes et al. 2017 Cas9 expression systems CaMV 35s promoter (for transient assays, not recommended for stable heritable events) Feng et al. 2013, Jiang et al. 2013, Shan et al. 2013 Species appropriate Ubiquitin promoter Miao et al. 2013, Wang et al. 2014, Peterson et al. 2016 Egg cell-specific (EC1.2) promoter Wang et al. 2015 SPL promoter Mao et al. 2016 LAT52 promoter Mao et al. 2016 DDT45 promoter Mao et al. 2016 Meiosis-specific (MGE1/MGE2/MGE3) promoter Eid et al. 2016 Cas9 modifications Codon optimization Fauser et al. 2014, Gao et al. 2015a, Jiang et al. 2013b, Miao et al. 2013, Shan et al. 2013 Paired nickases Fauser et al. 2014, Mikami et al. 2016, Schiml et al. 2014 dCas9 Lowder et al. 2015, Piatek et al. 2015 Heat shock treatment LeBlanc et al. 2018 Alternative endonuclease varieties SaCas9 Zhang et al. 2017, Steinert et al. 2015 St1Cas9 Steinert et al. 2015 SpCas9-HF1 Kleinstiver et al. 2016 eSpCas9(1.1) Slaymaker et al. 2016 HypaCas9 J.S. Chen et al. 2017 Cpf1 Xu et al. 2017, Kim et al. 2017 Cas13a Abudayyeh et al. 2017 Cas9 as a Genome-Editing Tool: General Considerations Target selection and the specificity of gRNAs RNA-mediated DNA homing is central to any Cas9 genome editing application, whether the goal is to knock out gene function, replace endogenous gene sequences or home Cas9 to DNA for other applications. For SpCas9, gRNA target sites are typically 19–20 nucleotides (nt) in length and are flanked on the 3' end by an NGG PAM site. Truncated gRNAs (tru-gRNAs) that are 17–18 nt, compared with the traditional 20 nt size, can increase specificity without compromising efficiency (Fu et al. 2014, Ren et al. 2014, Moreno-Mateos et al. 2015, Tsai et al. 2015). Initially tested in zebrafish, Drosophila and human cell lines, tru-gRNAs were recently used to induce mutations efficiently in Arabidopsis (Osakabe et al. 2016). Cas9 nuclease activity cuts double-stranded DNA at the –3 position relative to the PAM and is strand independent in Arabidopsis (T. Wang et al. 2014, Peterson et al. 2016). gRNAs bind to their target site by hybridizing to the antisense strand of the DNA target. Studies have shown that the GC content of the target site is a factor in editing efficiency and specificity. A GC content of 40–60% seems to be the preferred range in mammalian cell lines (Liu et al. 2016). In human cells, mismatches between gRNA and the target site are tolerated to a degree, leading to off-target Cas9 binding and cleavage events (Fu et al. 2013). The region most distal from the PAM, the tail region, is more permissive of mismatches, while the seed region (the 8–12 nt most proximal to the PAM) is critical for site recognition and also does not tolerate mismatches (Jinek et al. 2012, Liu et al. 2016) (Fig. 1). A high degree of homology within the seed region can result in off-target binding (Pattanayak et al. 2013). A favorable target site should differ from potential off-target sites by at least a few nucleotides, preferably in the seed region, to avoid the possibility of off-target editing. Online programs, such as CRISPR-P and CRISPR-Plant, can be used to design gRNAs specifically for a variety of fully sequenced plant genomes and are helpful in insuring that perfect target site matches do not exist elsewhere in the genome. Both programs use algorithms to analyze the intended target sites; CRISPR-P then identifies potential off-target sites throughout the genome (Lei et al. 2014), while CRISPR-Plant prioritizes target sites with the lowest probability of off-target editing (Xie et al. 2014). It should be noted that gRNA targeting programs identify off-targets based on extrapolated gRNA binding rules. Unbiased approaches to identify off-target events have been generated by several groups. Genome-wide, unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) works by tagging DSBs in live cells using blunt, double-stranded oligodeoxynucleotides (dsODNs) and then mapping these dsODNs via sequencing (Tsai et al. 2015). A similar method, direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (BLESS), maps DSBs using a barcode-containing biotinylated linker (Crosetto et al. 2013). Chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) using catalytically inactive Cas9 can also identify Cas9-binding sites, (Kuscu et al. 2014), but binding does not always correlate with editing (Wu et al. 2014, Tsai et al. 2015). To our knowledge, these methods have not been used to detect editing in plants, but theoretically could be applied to plant protoplasts (Shi et al. 2016). More importantly, unbiased approaches that enrich for Cas9 cleavage events have revealed that for SpCas9 there is little overlap between predicted off-target sites and true off-target sites (Tsai et al. 2015). As such there is still much to learn about target selection and much remains to be confirmed in plants. Fig. 1 View largeDownload slide Overview of the CRISPR-Cas9 system. The system consists of the gRNA (white), which guides the Cas9 nuclease to the genomic target site (pink). The genomic target region (purple indicator) is composed of 20 bp that are homologous to the gRNA (white) and a PAM sequence (green indicator). The tail region (orange indicator) of the genomic target, which is most distal from the PAM motif, is more permissive of mismatches, while the 8–12 nt seed region (blue indicator) most proximal to the PAM is critical for site recognition of the CRISPR/Cas9 complex. Cleavage occurs 3bp 5’ of the PAM sequence. Fig. 1 View largeDownload slide Overview of the CRISPR-Cas9 system. The system consists of the gRNA (white), which guides the Cas9 nuclease to the genomic target site (pink). The genomic target region (purple indicator) is composed of 20 bp that are homologous to the gRNA (white) and a PAM sequence (green indicator). The tail region (orange indicator) of the genomic target, which is most distal from the PAM motif, is more permissive of mismatches, while the 8–12 nt seed region (blue indicator) most proximal to the PAM is critical for site recognition of the CRISPR/Cas9 complex. Cleavage occurs 3bp 5’ of the PAM sequence. Despite these concerns, there is little actual off-target editing in Arabidopsis, tomato, maize, rice and tobacco when using SpCas9 compared with animals (Nekrasov et al. 2013, Feng et al. 2014, Gao et al. 2015, Svitashev et al. 2015, Woo et al. 2015, Ishizaki 2016, Pan et al. 2016, Peterson et al. 2016, Tang et al. 2016). In the most comprehensive study to date in Arabidopsis, pooled plants expressing 14 gRNAs targeting different genomic sites were analyzed by deep sequencing. On-target indel rates ranged from 33% to 92% of sequencing reads, but no off-target editing events were found elsewhere in the genome at predicted or other sites, confirming smaller scale studies (Peterson et al. 2016). In tomato, no off-target mutation events were found during editing of pathogen-related genes (Nekrasov et al. 2017), or when engineering tomato quantitative trait variation via targeted deletion of cis-regulatory elements (Rodríguez-Leal et al. 2017). Outside of Arabidopsis, the use of spCas9 has led to off-target events at low frequencies in some cases. Whole-genome sequencing experiments screening for off-target editing in rice revealed no large-scale off-target editing mutations in one study (Zhang et al. 2014), but low levels of off-target editing were detected at highly homologous sites that had a 1 bp mismatch to the target site outside of the seed region in other rice studies (Zhang et al. 2014, Xu et al. 2015). Similarly, off-target editing was found at a frequency <3.0% between sites, with a 1 bp mismatch at the ninth nucleotide in the seed region in wheat (Zhang et al. 2016) and off-targets can also be identified in maize but can be decreased with the use of ribonucleoprotein (RNP) complexes (Svitashev et al. 2016). The take-home message is that the specificity of SpCas9 is species specific and needs to be separately evaluated, preferably with unbiased off-target assessment methods, but off-target events are unlikely to be as common in plants as with animals. Some gRNAs do not work at all. In our lab, we estimate that about 5% of gRNAs do not function for reasons unknown (Peterson et al. 2016, Čermák et al. 2017). Chromatin accessibility and nucleosome occupancy can decrease the ability of Cas9 to bind DNA in animal studies (Kuscu et al. 2014, Wu et al. 2014, Horlbeck et al. 2016). However, despite its effect on binding efficiency, chromatin accessibility does not negate Cas9 double-stranded DNA cleavage per se (Hsu et al. 2013, Moreno-Mateos et al. 2015). We have yet to find a gene that cannot be targeted by Cas9 and suspect that chromatin accessibility is unlikely to be a critical limiting factor in most plant targeting experiments. Delivery of gRNAs and Cas9 to Plant Cells Transgene-based delivery systems Also central to all editing applications is the need to get gRNAs and Cas nucleases into plant cell nuclei. Agrobacterium-mediated transformation of transgene-delivered CRISPR components is the most common method and varies across species, including floral dip in Arabidopsis, callus and immature embryo transformation, leaf tissue transformation and regeneration, and hairy root transformation. The difference between Agrobacterium transformation methods can have a profound effect on downstream editing success, especially in gene knockout experiments. To summarize broadly, plants that use callus-based regeneration for transformation frequently yield transgenic plants homozygous for indels in target genes in the first generation with Cas9, while plants such as Arabidopsis, which use floral dip transformation methods, typically do not (discussed below). Recently a magnetic-based approach to pollen transformation, using transgenes loaded onto magnetic particles which are driven into pollen grains via magnetic field exposure, has been used to transform pollen in cotton and other species (Zhao et al. 2017). In theory this approach could be used to deliver CRISPR components and should be applicable to a broad range of species. Individual transgenic gRNA expression requires RNA polymerase III promoters, with U3 and U6 promoters being the most commonly used. gRNA transcription starts at an adenine or guanine nucleotide, respectively, for these promoters, but genomic target sites need not start with a guanine or adenine on the 5' end to be targeted, so long as the nucleotide is included in the promoter::gRNA construct (Shan et al. 2013, Ma et al. 2015). Generally, editing efficiency can be increased by using a U6 promoter native to the species being edited (Sun et al. 2015). However, in cases where the U6 promoter is similar enough, as with tomato and Arabidopsis, using the Arabidopsis U6 promoter may be sufficient (Pan et al. 2016). For multiplexing, gRNAs can be expressed in linear arrays of Pol III promoter::gRNA ‘units’ in a single transgene in a head to tail orientation, with 16 gRNA units being the most we have delivered to Arabidopsis in a single transgene to date (Z.L.N., unpublished). Alternatively, multiple gRNAs can be encoded in a single polycistronic mRNA which is then processed to individual units by cis-elements that allow processing and release of individual gRNAs. One advantage is that polycistronic RNAs allow the use of POL II promoters for transcription, permitting cell-specific expression in theory. Polycistronic systems include self-cleaving ribozymes (Gao and Zhao 2014), and also systems that depend on accessory enzymes for gRNA release. For example, the Csy4 endoribonuclease expressed with Cas9 can be used to process gRNAs that are cleavable by Csy4 (Tsai et al. 2014). Using the endogenous tRNA processing system, multiplex genome editing was successfully achieved in rice with efficiencies up to 100% (Xie et al. 2015). There are additional species-specific effects on gRNA expression. For example, expression of gRNAs was lower in Arabidopsis compared with rice, possibly as a result of gRNA silencing (Jiang et al. 2014, Ma et al. 2015). In mammals, higher gRNA expression levels have been correlated with increased off-target editing. If gRNA expression is too high, reduction can be accomplished by inclusion of a UUU stretch in the seed region, which may lead to partial transcriptional termination and lower expression levels (Wu et al. 2014). Given the higher degree of specificity in plants, it is unlikely that gRNA expression levels are a general concern. In order to facilitate DNA cleavage, all systems to date employ Cas9 containing nuclear targeting sequences. To drive expression of Cas9, many systems use a constitutive promoter, such as Cauliflower mosaic virus (CaMV) 35S, or monocot- or dicot-specific versions of Ubiquitin promoters (Feng et al. 2013, Jiang et al. 2013, Miao et al. 2013, Shan et al. 2013, . Wang et al. 2014, Peterson et al. 2016). For floral dip transformation methods in Arabidopsis, it is not recommended to use the 35S promoter as this will prevent Cas9 expression in meristematic and germline cells of the shoot, precluding heritable genetic changes from being passed to progeny. Promoters that are active in callus are critical for callus regeneration methods and may be tied to the highly efficient generation of editing events in the T1 generation in callus-regenerated species (see below). Expression of Cas9 from egg cell-specific promoters may be used to restrict editing to the egg cells and one-cell stage embryos (Wang et al. 2015). Additional germline-specific promoters for Cas9 have been tested in Arabidopsis, including the SPL promoter, which targets sporogenous cells and microsporocytes, the LAT52 promoter, which targets pollen cells, and the DDT45 promoter, which targets egg cells (Mao et al. 2016). DDT45 was capable of producing heritable mutations in the T1 generation (Mao et al. 2016). Meiosis-specific promoter-driven expression of Cas9 has also been used to restrict editing (Eid et al. 2016). It is critical to keep in mind that the choice of promoter has strong effects on the ability to recover and select mutants, particularly in gene knockout experiments. The different expression systems used in CRISPR/Cas9 editing of plant genomes are covered in more detail by Lowder et al., who provide an excellent summary of the topic (Lowder et al. 2016). Several groups have published finished or assembly-ready cloning systems (Lowder et al. 2015, Ma et al. 2015, Čermák et al. 2017). Provided any particular toolkit uses appropriate promoters and selection, and have proven to work in planta and recover heritable mutants, user preference should govern choice. However, when it comes to multiplexing with transgene delivery, the need to create linear arrays of gRNAs, either with their own promoter or from a single cleavable transcript, poses cloning challenges. Golden Gate and Gibson assembly methods offer significant advantages in flexibility and cost (Lowder et al. 2015, Ma et al. 2015) and have been able to assemble as many as 12 gRNAs into a single vector (Čermák et al. 2017). Gene synthesis of gRNA arrays, followed by blunt-end cloning assembly, have allowed for the creation of 14 gRNA arrays in a single transgene, but is more expensive and slower than Golden Gate assembly (Peterson et al. 2016). Non-transgene delivery systems Biolistic delivery of Cas9/gRNA constructs has been successfully used in rice (Shan et al. 2013). Biolistic delivery methods can have higher rates of incomplete incorporation of the Cas9 transgene upon transformation when compared with Agrobacterium-mediated transformation, as seen in soybean (Jacobs et al. 2015). However, biolistic delivery methods are superior to Agrobacterium-mediated transformation when the goal is gene insertion via HDR, possibly a reflection of the number of DNA molecules delivered (Svitashev et al. 2015). More recently, the same group demonstrated that a pre-assembled Cas9 protein–gRNA RNP complex can be successfully introduced into maize embryos via particle bombardment with higher specificity compared with a non-RNP treatment (Svitashev et al. 2016). RNPs have been used in protoplasts to regenerate edited lettuce (Woo et al. 2015). While originally limited to plants that could be regenerated via protoplasts, methods for RNP delivery to immature embryos are now established in wheat and maize (Svitashev et al. 2016, Liang et al. 2017). Alternatively, if a species is recalcitrant to protoplast regeneration, either transiently expressed CRISPR/Cas9 DNA (TECCDNA) or transiently expressed CRISPR/Cas9 in vitro transcripts (TECCRNAs) can be delivered to immature embryos. In wheat, this system has been successfully used to generate homozygous mutations in the T0 generation (Zhang et al. 2016). Compared with RNP delivery into immature embryos, TECCDNA is more efficient at inducing on-target mutations, although RNPs yield a better on-target to off-target editing ratio (Liang et al. 2017). Cas Nuclease Options SpCas9 has been extensively studied with the goal of improving function in animal systems. There are many available plant codon-optimized versions of SpCas9, some which are broadly optimized for plants in general (Li et al. 2013, Xu et al. 2014, Ma et al. 2015), others that are optimized specifically for monocots (Jiang et al. 2013) and still others that are specifically optimized for a particular species, such as rice, tobacco or Arabidopsis (Jiang et al. 2013, Miao et al. 2013, Shan et al. 2013, Fauser et al. 2014, Gao et al. 2015, Peterson et al. 2016), and may lead to higher expression levels and higher editing efficiencies in some cases (Li et al. 2013, Xu et al. 2014). As mentioned above, in some plants, wild-type SpCas9 is exquisitely specific to on-target editing and, even in plants where off-target events are seen, the rates are very low. However, protein engineering has been used to improve SpCas9 specificity to reduce off-target editing while maintaining on-target efficiency in human cells. The most specific of these newly designed variants are high-fidelity (SpCas9-HF1) (Kleinstiver et al. 2016), enhanced specificity [eSpCas9(1.1)] (Slaymaker et al. 2016) and hyper accurate Cas9 (HypaCas9) (J.S. Chen et al. 2017). Comprehensive comparisons of these variants have not been done in plants, but could help reduce off-target rates further in plant species with higher off-target rates. SpCas9 proteins with mutations in the RuvC or HNH catalytic sites result in nickase mutants that exclusively cut either the plus or minus DNA strand (Gasiunas et al. 2012). The use of a single nickase is suitable for inducing HDR, but not for inducing NHEJ, as demonstrated in Arabidopsis (Fauser et al. 2014). However, if two Cas9 nickases are directed by a pair of gRNAs targeting opposite strands of a genomic target, it is possible to generate DSBs to be repaired via NHEJ (Ran et al. 2013). Since two gRNAs are required to identify and bind to appropriate targets, this method greatly decreases off-target editing events (Ran et al. 2013). One drawback is that the specific positioning of the paired targets decreases the number of usable target sites. Paired nickases have been used successfully to edit Arabidopsis and rice (Schiml et al. 2014, Mikami et al. 2016). Editing efficiency can be improved through the optimization of the distance between two gRNAs, and by using nickases that generate either 5' or 3' overhangs, depending on whether NHEJ or HDR is favored (Mikami et al. 2016). Similarly, catalytically inactive Cas9 can be paired with the nuclease domain of the FokI restriction enzyme to create a dimer that will nick DNA, which increases specificity due to the Fok1 dimerization requirements (Guilinger et al. 2014, Tsai et al. 2014). There are many Cas9 orthologs and alternative CRISPR effector nucleases. Some variants recognize different PAM sequences, cut DNA in a different manner or even target RNA. However, these may come with different efficiencies and specificities, or are not as well characterized as SpCas9. For most experiments, NGG PAM sites are readily found in target genes and wild-type SpCas9 will suffice. For very specific targeting of small genes or DNA elements, having different PAM options can be advantageous. SpCas9 preferentially recognizes the PAM 5'-NGG-3' but it will also recognize NAG, NGA, NAA, NGT, NGC and NCG at much lower frequencies, which is probably not practical (Hsu et al. 2013, Tsai et al. 2015). However, engineered versions of SpCas9 exist that preferentially recognize alternative PAM sites, including NGCG, NGAN and NGCG (Kleinstiver et al. 2015). Orthologs, such as SaCas9 from Streptococcus aureus, recognize the PAM sequence NNGRRT (Ran et al. 2015), and can induce editing in Arabidopsis (Zhang et al. 2017). Cas9 varieties found in Streptococcus thermophilus and Brevibacillus laterosporus have also been used successfully in Arabidopsis and maize, respectively (Karvelis et al. 2015, Steinert et al. 2015). In addition to recognizing alternative PAMs, other versions of Cas9 are in some cases smaller in size than SpCas9, which may help in some delivery systems (Steinert et al. 2015). Additionally, multiple Cas9 systems can be used simultaneously in order to maximize the number of sites targeted (Zhang et al. 2017). Having alternatives to NGG PAM sites expands options; however, it remains unclear if these variants will be as specific as wild-type SpCas9 in plants in many cases. Higher off-target editing has been observed in Arabidopsis when using SaCas9, though this may be an artifact of using gRNA design programs optimized for SpCas9 (Zhang et al. 2017). Further species-specific work is necessary to make broad conclusions in most cases. Cpf1 (CRISPR from Prevotella and Francisella1) is a single RNA-guided endonuclease that recognizes the PAM TTN and generates staggered DSBs, unlike SpCas9 (Zetsche et al. 2015). In rice, the Cpf1 system has successfully been used to generate heritable mutations with no off-target editing (Xu et al. 2017). Cpf1 is a useful alternative to SpCas9 because it generates larger deletions than SpCas9, as observed in soybean and tobacco protoplasts (Kim et al. 2017). It is important to note that when using Cas9, a trans-acting in vitro transcribed or chemically synthesized cRNA is necessary to generate mutations, while Cpf1 is guided by a single cRNA without the need for a transacting cRNA to induce mutations (Kim et al. 2017, Xu et al. 2017). Cas13a derived from Leptotrichia wadei is an RNA-guided RNase shown to be effective in reducing target transcript accumulation by >50% in rice protoplasts (Abudayyeh et al. 2017). This exciting finding opens up the possibility of robust RNA-mediated RNA targeting in plants, although it remains to be seen how effective Cas13a will be in producing phenotypic knockdowns in whole plants. A General Comment on Efficiency In general, the editing efficiency and specificity of a particular approach must be evaluated at the level of stable plant generation in the species of interest. Extrapolation of rules derived from protoplasts and other species does not necessarily insure similar success levels across platforms. Most importantly, there is considerable variability in gRNA efficiency, which does not appear to change with expression system or Cas9 delivery method and is often hard to predict. Despite all the modifications and alternatives, for many applications SpCas9 will suffice and there may be no clear advantage for one system over another in efficiency or specificity. This is especially true in single gene mutation targeting in callus-regenerating plants, which typically yields high rates of homozygous mutant recovery, or in Arabidopsis when considering specificity. In those cases, the ease of use of the system and its record of validation in planta might be the strongest consideration. Recent work provides evidence that heat shock treatment of plants at 37°C for brief periods of time can enhance rates of Cas9 editing (LeBlanc et al. 2018). This effect is apparent in both somatic and germline tissues, where as much as 100-fold increases of editing rates are seen. This effect is linked to higher efficiency of Cas9 double-stranded DNA cleavage at 37°C. Excitingly, this simple treatment works in multiple species, suggesting that this may be an excellent tool to bolster editing experiments across systems. Considerations for Creating Loss-of-Function Gene Mutations One of the simplest uses of CRISPR in plants has been to generate heritable knockout mutants to assess gene function, but this approach has several considerations. One of the main considerations is whether to delete a gene entirely or create a frameshift mutant using a smaller indel. The majority of Cas9 indels occur at the –3 site to the PAM sequence in the protospacer sequence, where Cas9 cuts. In tomato, some mutations were reported to occur between the fourth and fifth base upstream of the PAM, 1 bp further than typically expected (Doudna and Charpentier 2014, Pan et al. 2016). A few cases of nucleotide substitutions have been noted in rice and Arabidopsis arising from repair (Feng et al. 2014, Zhang et al. 2014, Ishizaki 2016). For frameshift-induced indels, targeting the first exon closest to the initiator methionine can usually suffice to truncate function early. For instance, targeting the CORYNE gene with a single gRNA in Arabidopsis resulted in a truncation at amino acid 6 out of 402, a clear null (Nimchuk 2017). In some cases, collateral nonsense-mediated mRNA decay may accompany frameshift mutants (Copenhaver, personal communication). If two distinct gRNAs are used to target exons simultaneously, it can insure editing should one gRNA not work, and provide a broader collection of mutant alleles. However, if the Cas9 transgene is still functioning in a single editing site mutant, phenotypic revertants can arise when deletions and/or insertions at the second editing site restore the frame in rare cases (Z.L.N. unpublished). Typically indels are small, mostly single base pair changes across species (Feng et al. 2014, Zhang et al. 2014, Ma et al. 2015, Svitashev et al. 2015, Ishizaki 2016, Pan et al. 2016) (Fig. 2A). Larger deletions occur at increasingly lower frequencies and therefore to create a true complete deletion of a gene, dual gRNAs flanking the gene are typically required. Such large-scale deletions, or two-site deletions, can be generated when editing occurs at two nearby target sites simultaneously and endogenous repair mechanisms repair the DNA breaks by ligating the 3' end of one target with the 5' end of the other (Moreno-Mateos et al. 2015) (Fig. 2C). Two-site deletions have been successfully induced in rice, Arabidopsis, tobacco and tomato (Brooks et al. 2014, Feng et al. 2014, Zhou et al. 2014, Lowder et al. 2015, Ma et al. 2015, Xie et al. 2015, Gao et al. 2016, Mikami et al. 2016). Gene deletion frequencies differ strongly across protoplasts, callus and floral dip regeneration. Callus regeneration often gives rise to longer deletion events that occur between two gRNAs (Gao et al. 2016, Čermák et al. 2017, Rodríguez-Leal et al. 2017) while comparable deletions are recovered in T2 plants of Arabidopsis through floral dip at a low frequency; around 1% of plants in our hands (Z.L.N., personal observation) (Peterson et al. 2016). As such, PCR-based screening systems to enrich such plants will need to be performed, unless the phenotype is known and can be used to select mutant plants from the population. Fig. 2 View largeDownload slide CRISPR/Cas9 methods to alter gene function. The cartoon depicts various scenarios when using different Cas9 applications. (A) A double-stranded break at the target sequence activates the NHEJ pathway that results in the insertion or deletion of a random base at the site of repair, which causes a frameshift mutation. (B) Homology-directed repair mechanisms use homologous regions to rejoin cleaved DNA often with the introduction of donor DNA to create an intended modified DNA. (C) Cas9-induced large deletions are achieved by using multiple gRNAs flanking the gene of interest. Such large-scale deletions, or two-site deletions, can be generated when editing occurs at two nearby target sites simultaneously and endogenous repair mechanisms repair the DNA breaks by ligating the 3' end of one target with the 5' end of the other. (D) dCas9 acts as an artificial transcription factor when fused with a transcriptional activator or repressor. (E) A base editor, consisting of dCas9 and a cytidine deaminase converts C to U in a targeted base pair. The DNA repair pathway converts the resulting U:G mismatch to T:A. Fig. 2 View largeDownload slide CRISPR/Cas9 methods to alter gene function. The cartoon depicts various scenarios when using different Cas9 applications. (A) A double-stranded break at the target sequence activates the NHEJ pathway that results in the insertion or deletion of a random base at the site of repair, which causes a frameshift mutation. (B) Homology-directed repair mechanisms use homologous regions to rejoin cleaved DNA often with the introduction of donor DNA to create an intended modified DNA. (C) Cas9-induced large deletions are achieved by using multiple gRNAs flanking the gene of interest. Such large-scale deletions, or two-site deletions, can be generated when editing occurs at two nearby target sites simultaneously and endogenous repair mechanisms repair the DNA breaks by ligating the 3' end of one target with the 5' end of the other. (D) dCas9 acts as an artificial transcription factor when fused with a transcriptional activator or repressor. (E) A base editor, consisting of dCas9 and a cytidine deaminase converts C to U in a targeted base pair. The DNA repair pathway converts the resulting U:G mismatch to T:A. Plant Phenotypes with CRISPR/Cas9: Advantages and Challenges When using CRISPR/Cas9, different copies of a targeted gene may be mutated at different times. Depending on the Cas9 delivery system, this can occur at different stages and provides opportunities and challenges for isolating plants. When both copies of the targeted gene are mutated in an embryogenic cell before the initial division, the genotype may be homozygous, if the mutations are of the same nature, or biallelic, if two different mutations occurred in the two gene copies (Zhang et al. 2014). In callus-regenerated plant species, including tomato (Brooks et al. 2014, Pan et al. 2016), rice (Xu et al. 2015, Ishizaki 2016), lettuce (Woo et al. 2015) and wheat (Zhang et al. 2016), it is easy to detect homozygous biallelic mutations in T0 plants and with a high editing efficiency. Arabidopsis T1 mutants display an extremely low frequency of homozygous mutations in the first generation, even with germline-specific Cas9 promoters, with homozygous mutants recovering an increasing frequency in later generations (Fauser et al. 2014, Feng et al. 2014). In many cases, this provides a clear advantage for callus regeneration, except if the gene targeted is lethal, sterile or severely developmentally compromised when mutant. In these cases, recovery of heterozygous events may be necessary. Once editing has occurred or is inherited stably in germline cells, gRNA target sites will not re-cleave and are fixed. However, in plants not stably mutant, and still carrying Cas9-gRNA transgenes, considerable sectoring and chimerism in somatic cells is expected if Cas9 is expressed from a constitutive promoter (Fauser et al. 2014, Feng et al. 2014, Peterson et al. 2016). This occurs in first-generation transgenic plants, but also in subsequent generations (Fauser et al. 2014, Feng et al. 2014). Chimerism and mosaicism, without fixed heritable mutations, by themselves can give rise to phenotypes in plants when using constitutive Cas9 expression and to unpredictable phenotype ratios, often varying between transgenic lines (Zhu et al. 2014, Ishizaki 2016, Pan et al. 2016). Mosaic-induced phenotypes can arise in some cases at a very high rate even in T1 plants (Fig. 3). As such, phenotype alone does not insure plants are stable homozygous mutants. Fixed, homozygous mutants must be confirmed by individual plant sequencing and confirming Cas9 absence by segregating away Cas9 transgenes. In some cases, mosaicism can be advantageous, allowing a rapid estimate of what relevant phenotypes might be, or result in plants that are intermediate with null mutants and may help assign gene function. In other cases, sectoring can occur that may help guide homozygous mutant recovery. For instance, targeting of CORYNE in Arabidopsis gave rise to a single branch with all mutant flowers that arose from a stably mutated biallelic sector that propagated clonally. Collection of seed from that branch yielded all fixed mutants in the next generation, many Cas9 free, and two different alleles (Nimchuk 2017). In other cases, as noted above, strong mosaicism may be phenotypically deleterious, and germline editing can be used to identify heterozygous mutants in the first generation using promoters restricted to the germline to drive Cas9. Mosaicism can also be avoided by using pre-assembled CRISPR/Cas9 RNPs since they cleave chromosomal target sites immediately upon transfection and are presumably degraded by endogenous proteases within the cells over time (Woo et al. 2015). Fig. 3 View largeDownload slide Ubiquitous Cas9/gRNA expression can yield mosaic target gene editing and intermediate phenotypes. Null mutations in the WUSCHEL (WUS) gene in Arabidopsis severely compromise shoot growth and strongly reduce flower formation, and prevent fruit development (right). In contrast, mosaic editing of WUS with ubiquitously expressed Cas9 and WUS-targeting gRNA results in intermediate phenotypes displaying robust shoot growth, flower formation, but with missing floral organs at high frequencies (60% of transgenic plants). Left: close up of wild-type and Cas9/WUS gRNA-targeted inflorescences to show detail. Yellow arrows, mature fruit derived from individual flowers in the wild type; blue arrow, flower lacking fruit formation. Note the lack of flower development on wus null mutants. Fig. 3 View largeDownload slide Ubiquitous Cas9/gRNA expression can yield mosaic target gene editing and intermediate phenotypes. Null mutations in the WUSCHEL (WUS) gene in Arabidopsis severely compromise shoot growth and strongly reduce flower formation, and prevent fruit development (right). In contrast, mosaic editing of WUS with ubiquitously expressed Cas9 and WUS-targeting gRNA results in intermediate phenotypes displaying robust shoot growth, flower formation, but with missing floral organs at high frequencies (60% of transgenic plants). Left: close up of wild-type and Cas9/WUS gRNA-targeted inflorescences to show detail. Yellow arrows, mature fruit derived from individual flowers in the wild type; blue arrow, flower lacking fruit formation. Note the lack of flower development on wus null mutants. Ultimately it is necessary to sequence genes, and confirm phenotypes in plants lacking Cas9 transgenes by genetic segregation, before concluding that phenotypes are associated with heritable mutations. PCR and/or drug selection to determine T-DNA presence in T2 generations is a relatively straightforward approach. Seed-expressed mCherry cassettes on Cas9 transgenes can allow for the rapid identification of Cas9-free plants in the T2 generation (Gao et al. 2016). Screening for mutations can be done by direct sequencing, and this might be the easiest method if an obvious or expected phenotype arises in T2 plants in Arabidopsis, and is standard in T0 callus regeneration systems where homozygous mutant recovery is high and plant numbers are low. However, to detect segregating T2 events when phenotypes are unknown, or in higher order multiplex events, or rare T1 events in Arabidopsis, a mutational screening strategy might be desirable. When multiplexing in Arabidopsis, the ability to recover higher order T2 mutants depends on the gene editing efficiency of each of the gRNAs. As such, recovery of higher order mutants and inheritance patterns can be highly variable, thus a good screening strategy is advisable. gRNAs that potentially disrupt an endogenous restriction enzyme site can be designed and mutants can be identified using a restriction fragment length polymorphism assay (Nekrasov et al. 2013, Shan et al. 2013, Kim et al. 2014). However, this approach limits gRNA site selection options. Diagnostic restriction sites can be created using primers that incorporate nucleotide changes, similar to the derived cleaved amplified polymorphic sequences (dCAPS) (Neff et al. 1998) used with missense mutations, and mutants can be identified on this basis (Endo et al. 2015, Nimchuk 2017). This approach has been recently automated by the design of web-based programs that can identify primers that create diagnostic restrictions sites (indCAPS) (Hodgens et al. 2017). Critically, this program tolerates indels, unlike standard dCAPS identification programs. The T7 endonuclease I assay and Surveyor system can be used to identify mutations based on heteroduplex mismatches between wild-type and mutant DNA sequences (Qiu et al. 2004, Kim et al. 2009, Xie and Yang 2013, Y. Wang et al. 2014); however, these assays are more costly and require additional PCR and reaction steps, and are not able to distinguish between wild-type and biallelic homozygous mutations or biallelic and monoallelic heterozygous mutants (Kim et al. 2014). Heteroduplex DNA formation can also be used in melting curve analysis to identify indel events (Denbow et al. 2017). Since homoduplex DNA migrates at a faster rate than heteroduplex DNA due to the open angle located between the matched and mismatched heteroduplex strands caused by mutation, this feature can also be used to detect editing visually, using polyacrylamide gel electrophoresis (PAGE) (Zhu et al. 2014). In rice, the PAGE assay has been modified to use single-stranded DNA since it offers the advantages of higher precision and the ability to distinguish homozygous mutants from the wild type (Zheng et al. 2016). Alternatively, if larger deletions are expected, then PCR products can be run on an appropriate percentage agarose gel, or even polyacrylamide gels. However, these results can be easily misinterpreted because small indels can yield wild-type-appearing bands and, occasionally, the slower migrating heteroduplex products will mistakenly appear as insertions (Brooks et al. 2014). In our hands, indCAPS and direct sequencing are the most robust and cost-effective way to screen for mutations, although plate-based Sanger sequencing of T2 plants can also provide a rapid and complete approach if time is more of an issue and cost is not. Multiple, independently derived alleles usually insure that phenotypes are associated with specific gene mutations, and complementation experiments should be performed if necessary. However, for extreme higher order multiplex mutations, which can be upwards of 10 genes in some cases and take several generations, technical and financial restrictions make complete independent mutation set identification and comprehensive complementation experiments challenging. Many genes important for crop traits are derived from mutations that alter but do not eliminate gene function, often resulting in cis regulatory mutations. A different approach for altering gene function via CRISPR is the targeting of wild-type Cas9 to endogenous promoter elements, rather than the coding region of the gene of interest itself, to create indels in promoter sequences that alter gene expression and function. Specific promoter elements, if known, can be targeted, but promoter sequences can also be randomly mutated in a blind fashion by exploiting the ability to multiplex with Cas9. This powerful random approach was recently demonstrated in tomato using suites of gRNAs spaced across different promoter sequences. This resulted in populations of plants with a continuum of non-null phenotypes arising from a variety of promoter deletion/insertion events between gRNAs which often altered target gene expression levels. This exciting promoter targeting approach is likely to be highly advantageous for crop breeding as it can rapidly create a broad range of trait variants that can be exploited for breeding (Rodríguez-Leal et al. 2017). Repairing and Replacing Genes with CRISPR in Plants The generation of mutations by CRISPR is dependent on the ability of the host species to repair DNA breaks. The most frequently observed repair mechanism is NHEJ, which generates unpredictable indels at specific target sites. A less frequently used repair mechanism is HDR, which can replace a stretch of sequence with an externally supplied sequence of choice, also known as gene repair. Repair via HDR is advantageous for creating knock-in mutants, which include applications such as substitution of a promoter for increased expression of a stress response gene (Shi et al. 2017) and the modification of herbicide resistance genes (Svitashev et al. 2015, Endo et al. 2016, Sun et al. 2016). While CRISPR editing in plants has predominantly focused on NHEJ events, multiple species have been successfully edited via HDR, including tomato (Čermák et al. 2015), rice (Endo et al. 2016, Sun et al. 2016, Wang et al. 2017), wheat (Gil-Humanes et al. 2017), maize (Svitashev et al. 2015, Shi et al. 2017), Arabidopsis (Zhao et al. 2016) and soybean (Li et al. 2015), among others. Generally, the editing efficiency of gene targeting is significantly lower for indel generation, with efficiencies typically <1% (Svitashev et al. 2015, Endo et al. 2016). This may suffice if a clear phenotype or selection for the replacement is available, but in most cases these rates would necessitate considerable screening of plants. NHEJ repair pathways compete with HDR and, in plants, there appears to be a strong bias towards NHEJ, complicating gene replacement methods (Qi et al. 2013). Additional barriers include the temporal delivery of the components to dividing cells, where the HDR pathway predominantly functions, and the complexity of the components needed for gene repair and replacement. Repairing or replacing genes through HDR requires Cas9, CRISPR target site(s) flanking, or within, the desired insertion site, and a DNA sequence, often called the ‘donor DNA’, to be inserted (Fig. 2B). The sequence to be inserted is typically flanked on the 5' and 3' ends, with homology arms, or sequences that are homologous to the native genomic region, upstream and downstream of the insertion site. Homology arms from 46 bp to 1 kb in length have been used successfully (Li et al. 2015, Svitashev et al. 2015, Sun et al. 2016, Gil-Humanes et al. 2017, Shi et al. 2017, Wang et al. 2017). While these additional DNA sequences are required for gene replacement, they further complicate the transformation process, as the efficiency of Agrobacterium-mediated transformation drops with an increase in the size of the vector or may lead to unwanted insertion of vector DNA. Methods circumventing these particular challenges include: transformation through particle bombardment (Li et al. 2015, Svitashev et al. 2015, Sun et al. 2016, Shi et al. 2017), sequential Agrobacterium transformation (Endo et al. 2016) and Agrobacterium-mediated transformation with a geminiviral vector (Čermák et al. 2015, Gil-Humanes et al. 2017, Wang et al. 2017).The use of geminiviral vectors increases editing efficiency, making this technique more feasible (Gil-Humanes et al. 2017, Wang et al. 2017). There is considerable interest in increasing the efficiency of gene repair and replacement, and strategies promoting HDR have been explored extensively in animals. These strategies include reducing the effectiveness of NHEJ by genetic or pharmacological targeting of NHEJ pathway components, using Cas effectors or combinations of nickases to avoid creation of a DSB, and targeting dividing cell populations (Pawelczak et al. 2017). Genetically manipulating NHEJ in Arabidopsis has increased gene efficiencies when using zinc finger nucleases. These mutants have not been used in conjunction with CRISPR/Cas9, but the technique shows promise for increasing the efficiency of gene repair and replacement in plants (Li et al. 2007, Qi et al. 2013, Zhang et al. 2017). Sigma-Aldrich maintains a nice collection of information on drugs used to bias HDR in CRISPR applications, but to our knowledge most of these have not been characterized in plants. Homing Applications of Cas Nucleases in Plants The RNA-based DNA homing ability of Cas9 independent of Cas9 nuclease activity (dead Cas9, dCas9) can be used to target dCas9 fusion proteins to DNA in a sequence-specific manner for various applications (Didovyk et al. 2016). Efficient targeted gene activation and repression have been achieved by the fusion of activation or repressor domains, respectively, to dCas9 (Fig. 2D) (Lowder et al. 2015, Piatek et al. 2015, Vazquez-Vilar et al. 2016). For single gene overexpression, it may be easier and more efficient to drive gene expression from heterologous promoters, but this approach could be particularly advantageous for multiplexed gene expression regulation, especially for functionally related genes in biochemical pathways. For single gene overexpression, it may be easiest to drive gene expression from heterologous promoters instead. The fusion of dCas9 to VP64 or VPR transcriptional activator domains increases transcriptional activation of endogenous targets (Chavez et al. 2015, Čermák et al. 2017) (Fig. 2C). The CRISPR–Cpf1 system has also been used for targeted gene repression in Arabidopsis with transcriptional reductions of up to 10-fold (Tang et al. 2017). In addition to creating DSBs, Cas9 can also be exploited to mutate nucleotides, or base edit. In base editing experiments, dCas9 fusions are made with cytosine deaminase domains allowing gRNA-mediated targeting of the fusion protein to genomic DNA and the creation of nucleotide substitutions rather than indels (Hess et al. 2017) (Fig. 2E). At the targeted DNA sites, cytosine deaminase acts upon cytosines near or within the targeted DNA, resulting in heritable conversions of C–G pairs to A–T pairs. The base editing approach has been used in several plant species and could be useful for creating allelic series in target genes or for protein structure–function analysis (Y. Chen et al. 2017, Shimatani et al. 2017, Zong et al. 2017). In animals, there is considerable interest in improving the rates of base editing and determining which cytosines in a targeted DNA region will be modified, as base editors tend to act on cytosines near or in targeted regions in an indiscriminant manner. Exciting recent work has resulted in the creation of adenine base editors, allowing A–T to G–C conversions (Gaudelli et al. 2017). The use of these editors in plants remains to be explored but opens up several new avenues for genome editing. Discussion The CRISPR/Cas9 system is the most recent and exciting addition to the genome editing toolbox because of its simplicity and successful application. The wide array of CRISPR/Cas9 optimizations allows the system to be used for multiple purposes with increasing efficiency and specificity. Newer technologies enabling epigenetic and transcriptional regulation should complement more traditional methods and could be used to tune or multiplex plant gene expression with unprecedented control. The ability to base edit in plants is in its infancy but, considering the potential applications for targeted in vivo substitution mutagenesis, represents an exciting opportunity. The massive increase in CRISPR systems and tools has begun to change what we can do with model plants. There is likely to be a resurgence in defining plant gene function, particularly for very small genes, discrete DNA elements and highly redundant gene families arising from this technology. The technology has even changed what a model system is, and should revolutionize comparative plant evolution studies with its ability to define gene function in traditionally non-model plants. Lastly, the ability to precisely target and modify gene functions in crops should provide a key tool in the hunt for new traits to meet the challenges facing global food production. Given the broad interest and push in CRISPR systems, new approaches to increasing efficiency, specificity, heritability and new CRISPR variants and applications are inevitable in the near future. It is an exciting time to be involved in plant research. Funding This work was supported by the National Science Foundation [grant No. IOS-1546837 to Z.L.N.]. Acknowledgments We would like to extend an apology to colleagues whose research we were unable to discuss due to length limitations. We thank Drs. Joe Kieber, Carly Sjogren and Ashley Crook for comments on the manuscript. Disclosures The authors have no conflict of interest to declare. References Abudayyeh O.O. , Gootenberg J.S. , Essletzbichler P. , Han S. , Joung J. , Belanto J.J. , et al. ( 2017 ) RNA targeting with CRISPR–Cas13 . Nature 550 : 280 – 284 . Google Scholar CrossRef Search ADS PubMed Anders C. , Bargsten K. , Jinek M. ( 2016 ) Structural plasticity of PAM recognition by engineered variants of the RNA-guided endonuclease Cas9 . Mol. Cell 61 : 895 – 902 . Google Scholar CrossRef Search ADS PubMed Barrangou R. , Fremaux C. , Deveau H. , Richards M. , Boyaval P. , Moineau S. , et al. ( 2007 ) CRISPR provides acquired resistance against viruses in prokaryotes . 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Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations CAPS cleaved amplified polymorphic sequence Cpf1 CRISPR from Prevotella and Francisella CRISPR/Cas9 clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 crRNA CRISPR RNA DSB double-stranded break dsODN double-stranded oligodeoxynucleotide gRNA guide RNA HDR homology-directed repair NHEJ non-homologous end joining PAM protospacer adjacent motif RNP ribonucleoprotein SaCas9 Streptococcus aureus Cas9 SpCas9 Streptococcus pyogenes Cas9 TECCDNA transiently expressed CRISPR/Cas9 DNA TECCRNA transiently expressed CRISPR/Cas9 RNA tru-gRNA truncated guide RNA tracrRNA trans-activating CRISPR RNA © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Plant and Cell PhysiologyOxford University Press

Published: Aug 1, 2018

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