TY - JOUR
AU - Su,, Yi-Hsien
AB - Abstract Studies on the gene regulatory networks (GRNs) of sea urchin embryos have provided a basic understanding of the molecular mechanisms controlling animal development. The causal links in GRNs have been verified experimentally through perturbation of gene functions. Microinjection of antisense morpholino oligonucleotides (MOs) into the egg is the most widely used approach for gene knockdown in sea urchin embryos. The modification of MOs into a membrane-permeable form (vivo-MOs) has allowed gene knockdown at later developmental stages. Recent advances in genome editing tools, such as zinc-finger nucleases, transcription activator-like effector-based nucleases and the clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) system, have provided methods for gene knockout in sea urchins. Here, we review the use of vivo-MOs and genome editing tools in sea urchin studies since the publication of its genome in 2006. Various applications of the CRISPR/Cas9 system and their potential in studying sea urchin development are also discussed. These new tools will provide more sophisticated experimental methods for studying sea urchin development. : morpholino, genome editing, CRISPR/Cas9, sea urchin Introduction Sea urchins have been a research model in developmental biology for over a century [1]. In recent decades, studies on the molecular mechanisms controlling the early developmental processes of the embryos have assembled developmental gene regulatory networks (GRNs) that elucidate causal links between regulatory factors and genomic sequences [2, 3]. These regulatory factors, including transcription factors and signaling molecules, are activated initially by asymmetrically distributed or differentially activated maternal factors present in the egg. The hierarchical activation of regulatory factors then leads to the expression of differentiation gene batteries that control cell differentiation, movement and various developmental processes. Functional perturbation of these regulatory factors followed by measurement of the effects on the potential target genes has enabled the establishment of epistatic relationships required for the construction of experimental GRN models. Several approaches have been used to perturb gene function in sea urchin embryos, including pharmacological treatment, injections of mRNAs encoding dominant-negative forms, morpholino oligonucleotide (MO) injection and, recently, targeted genome editing. Pharmacological treatments are commonly used for interfering with signaling pathways [4–10]. By directly adding antagonists or agonists into embryo cultures, signaling can be perturbed and precisely controlled during any developmental interval. Notably, most of these drugs target signaling transduction processes elicited by multiple signaling ligands and thus are not best suited for inferring the functions of individual genes. To achieve gene-specific perturbations, dominant-negative mRNA and MOs can be introduced into the embryos via microinjection. Dominant-negative forms of regulatory factors, in which their functional domains are either mutated/deleted or replaced with a repressive domain, are able to compete with the endogenous factors, thus inhibiting their regulatory functions [7, 11–16]. MOs, which are usually ribonucleotide oligos of 25 bases in length, can be designed to inhibit translation or mRNA splicing by binding to the complementary sequences in transcripts. The knockdown effect mediated by MOs can persist for up to a few days, making MOs a routinely used perturbation agent for constructing GRN models [7, 17–21]. Delivery of these gene-specific perturbation reagents is commonly achieved through microinjection in zygotes. Therefore, the perturbations are limited by the scale at which microinjection can be performed, and they lack spatial and temporal control. Regulatory genes are often expressed repeatedly in different cell types during development; the typical perturbation method thus becomes an issue for studying later functions of regulatory factors without interfering with their early roles. Only in recent years has delivery of MOs into sea urchin embryos at later stages become possible. Vivo-MOs, which are modified MOs linked to a molecular transporter with eight guanidinium head groups, have been shown to be effective in penetrating cell membranes [22]. Therefore, vivo-MOs can be added into sea urchin cultures at the chosen developmental stage to achieve stage-specific perturbation [5, 23]. Recent advances in genome editing technologies have opened an additional avenue for gene-specific perturbation in sea urchin embryos. Three major families of targeted genome editing systems, ZFNs, transcription activator-like effector-based nucleases (TALENs) and clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9), have been applied to disrupt functions of sea urchin genes by introducing mutations into the coding sequences [24–28]. By targeting the regulatory genome, which is often located in the noncoding regions, these editing techniques also allow researchers to study the ‘upstream’ control mechanisms of gene expression—a critical aspect for network verification—in its native genomic environment. In this review, we focus on functional perturbations using vivo-MOs and genome editing techniques, intending to provide a summary of advancement in functional perturbation approaches in sea urchin developmental studies since its genome was published in 2006. The well-established approaches of injecting mRNA and regular MOs have been reviewed elsewhere [21, 29] and are not covered here. Vivo-antisense MOs Vivo-MOs contain two functional compartments: a morpholino oligo, ensuring the targeting specificity, and a covalently linked octa-guanidine dendrimer for membrane penetration. As with MOs, vivo-MOs can be designed to block translation, by binding to start codons, or to interfere with mRNA splicing by targeting intron–exon junctions. However, vivo-MOs have not been widely used in sea urchins because of two major limitations. First, the solubility of vivo-MOs in seawater limits the concentrations one can use when vivo-MOs are directly added to sea urchin cultures. To improve the solubility, vivo-MO stock solutions can be incubated at 65°C before being diluted in seawater, and the working solutions are then vortexed thoroughly before mixing with cultures. While solubility is sequence dependent, using vivo-MOs at over 20 µM often results in precipitates in seawater that affect embryogenesis. Second, in our experience, vivo-MOs are generally more toxic than regular MOs. Several tests are recommended for the use of vivo-MOs in sea urchin studies (Supplementary Figure S1). A pilot toxicity test is used to find concentrations that do not result in severe mortality. The efficacies of vivo-MOs are then validated using similar approaches as for regular MOs. For example, translation-blocking vivo-MOs are tested by injecting sea urchin zygotes with synthetic mRNA containing the MO target site upstream of an open reading frame encoding green fluorescent protein (GFP), and then the embryos are incubated with the corresponding vivo-MO. An effective vivo-MO is verified based on its ability to decrease the GFP signal. For splice-blocking vivo-MOs, reverse transcription polymerase chain reaction analyses would reveal aberrant splice variants in treated embryos. To avoid unnecessarily lengthy treatments, embryos are treated with the vivo-MO for different durations to identify the effective time windows. Two studies have used vivo-MOs with sequences of regular MOs that are known to be effective in the sea urchin Strongylocentrotus purpuratus [5, 23]. Translation-blocking vivo-MOs against nodal and bmp2/4 are effective when directly added to embryo cultures at 5 µM [5]. Vivo-MOs targeting the skeletogenic genes P16 [30] and P58b [31], by blocking translation and splicing, respectively, are also effective either by addition to cultures at 15 µM or injection into juvenile rudiment tissues at 20 µM in injection solutions [23]. With the advance of vivo-MOs, functional perturbations at later developmental stages of sea urchins have become feasible. Other approaches for gene knockdowns at later stages include the use of regular MOs with Endo-Porter (GeneTools), a novel peptide that delivers substances into cells through an endocytosis-mediated process [32], and Photo-MOs that can switch gene expression on or off with light [33]. However, these methods have not been applied in sea urchins, and their effectiveness remains unclear. We anticipate that these gene-specific functional perturbations will be important for understanding molecular mechanisms controlling sea urchin development beyond embryonic stages. Gene perturbation by targeted genome editing in sea urchins Targeted genome editing allows gene-specific functional perturbations at the DNA level. Recent advances in genome editing technologies, including ZFNs, TALENs and CRISPR/Cas9, have provided useful tools to generate gene knockouts in sea urchin systems. Based on the use of engineered nucleases brought to specific DNA target sites, all three methods introduce DNA double-stranded breaks (DSBs) followed by DNA repair through homology-directed repair (HDR) or nonhomologous end joining (NHEJ) (Figure 1). Specific mutations or insertions can be introduced by HDR, in which an exogenous DNA template is required for recombination at the cleavage sites. In the absence of a DNA template, DSBs are typically repaired by the error-prone NHEJ pathway, which uses single-stranded overhangs on the ends of the breaks to guide repair. NHEJ repairs the ends by directly ligating the compatible overhangs using the DNA ligase IV/Xrcc4 complex and is more efficient than HDR. When the overhangs are not compatible, NHEJ relies on other processing factors, such as polymerases and nucleases, to either extend or truncate the overhangs for ligation. Because of these additional processing steps, NHEJ often leads to random insertions or deletions (indels) around cleavage sites. When DSBs occur in the coding region of a gene, these editing events usually cause frameshifts, thus creating functionally inactive gene products. Alternatively, indels occurring in the regulatory sequences, such as enhancers and promoters, can lead to disruption of gene transcription. Both types of cases can be used for loss-of-function studies. Figure 1: Open in new tabDownload slide Endogenous DNA repair mechanisms. DNA DSBs can be repaired via two different pathways. The NHEJ pathway imperfectly repairs the break and creates small indels (red). The indels often result in frameshifts and/or a premature stop codon in coding regions or disruption of regulatory sites in noncoding regions. HDR requires a donor template for homologous recombination, thus resulting in a precise sequence modification (green). (A colour version of this figure is available online at: https://academic.oup.com/bfg) Figure 1: Open in new tabDownload slide Endogenous DNA repair mechanisms. DNA DSBs can be repaired via two different pathways. The NHEJ pathway imperfectly repairs the break and creates small indels (red). The indels often result in frameshifts and/or a premature stop codon in coding regions or disruption of regulatory sites in noncoding regions. HDR requires a donor template for homologous recombination, thus resulting in a precise sequence modification (green). (A colour version of this figure is available online at: https://academic.oup.com/bfg) Zinc-finger nucleases Zinc-finger nuclease (ZFN) genome editing requires a pair of ZFNs, each containing a customized array of zinc-finger binding domains that bind to specific DNA sequences and the nuclease domain of the restriction enzyme FokI (Figure 2A). An individual array contains three to six zinc-finger repeats, each recognizing a unique DNA triplet, and targets 9–18 bases of DNA sequence [34]. When two ZFNs are both recruited to their binding sites, the two nuclease domains dimerize and generate a DSB. The direction in which two ZFNs are orientated at the target site is important: dimerization occurs only when two ZFNs bind opposite strands of DNA with their C-termini in physical proximity. Because it is zinc-finger binding domains that dictate the DNA-binding ability of ZFNs, screening for zinc-finger arrays that bind desired sequences is crucial. Various strategies have been developed for this purpose, including modular assembly and selection-based methods. The modular assembly approach involves the use of a preselected library of zinc-finger modules and by combining these modules, based on their known specificity, ZFNs are constructed to recognize the DNA target of interest [35–37]. Either phage display or cellular selection systems are involved in the selection-based approaches [38–40]. Figure 2: Open in new tabDownload slide Schematics of ZFNs, TALENs and CRISPR/Cas9. (A) The DNA-binding domains of ZFNs consist of three to six zinc-finger domains; each recognizes 3 bp of DNA. Two FokI domains dimerize and cut the spacer sequence flanked by the two target sites. (B) The DNA-binding domains of TALENs consist of an array of TALE repeats; each recognizes a single-base nucleotide. The RVD in each TALE repeat accounts for the binding specificity; its composition is indicated. (C) The CRISPR/Cas9 system requires the Cas9 nuclease and a gRNA. Specificity is achieved through complementary binding of the gRNA to the target sequence and a specific three-nucleotide ‘PAM’ sequence. (A colour version of this figure is available online at: https://academic.oup.com/bfg) Figure 2: Open in new tabDownload slide Schematics of ZFNs, TALENs and CRISPR/Cas9. (A) The DNA-binding domains of ZFNs consist of three to six zinc-finger domains; each recognizes 3 bp of DNA. Two FokI domains dimerize and cut the spacer sequence flanked by the two target sites. (B) The DNA-binding domains of TALENs consist of an array of TALE repeats; each recognizes a single-base nucleotide. The RVD in each TALE repeat accounts for the binding specificity; its composition is indicated. (C) The CRISPR/Cas9 system requires the Cas9 nuclease and a gRNA. Specificity is achieved through complementary binding of the gRNA to the target sequence and a specific three-nucleotide ‘PAM’ sequence. (A colour version of this figure is available online at: https://academic.oup.com/bfg) ZFNs were the first genome editing approach tested in sea urchins [26]. Ochiai et al. have screened a bacterial one-hybrid system using zinc-finger randomized libraries and a mammalian cell culture assay to select for ZFNs targeting the hesC gene in the sea urchin Hemicentrotus pulcherrimus (Figure 3A). HesC functions as a repressor for genes responsible for the specification of primary mesenchymal cells (PMCs) [42]. Injections of the ZFNs targeting hesC upregulated the delta gene, which is one of the known target genes of HesC [42]. Sequence analyses revealed that 44% of the polymerase chain reaction (PCR) clones amplified from the injected embryos contained indels at the target site that resulted in frameshifts. The timing of the ZFN-mediated mutagenesis was estimated to have occurred between the eight-cell and unhatched blastula stages. Figure 3: Open in new tabDownload slide Synthesis of functional modules of the genome editing tools. (A) A commonly used approach for synthesizing ZFNs requires two rounds of bacterial one-hybrid selections (adapted from [41]), first to screen for individual zinc-finger that recognizes a DNA triplet (a) and then to screen for arrays of zinc-fingers recognizing a specific target site (b). ZFNs are then generated by fusing the arrays with a FokI domain. Functional ZFNs can be verified using a SSA assay in mammalian cells, where activated ZFNs create DSBs at the reporter construct and induce DNA repair that produces an active luciferase gene (c). (B) Assembly of TALE repeats using the Golden Gate cloning strategy. A pool of TALEs, each having a specific RVD, is digested with BsaI and ligated together to form TALE repeats. (C) Synthesis of CRISPR/Cas9 functional modules. Cas9 mRNA and gRNA can be synthesized through in vitro transcription. Alternatively, Cas9 protein and customized gRNAs are commercially available. (A colour version of this figure is available online at: https://academic.oup.com/bfg) Figure 3: Open in new tabDownload slide Synthesis of functional modules of the genome editing tools. (A) A commonly used approach for synthesizing ZFNs requires two rounds of bacterial one-hybrid selections (adapted from [41]), first to screen for individual zinc-finger that recognizes a DNA triplet (a) and then to screen for arrays of zinc-fingers recognizing a specific target site (b). ZFNs are then generated by fusing the arrays with a FokI domain. Functional ZFNs can be verified using a SSA assay in mammalian cells, where activated ZFNs create DSBs at the reporter construct and induce DNA repair that produces an active luciferase gene (c). (B) Assembly of TALE repeats using the Golden Gate cloning strategy. A pool of TALEs, each having a specific RVD, is digested with BsaI and ligated together to form TALE repeats. (C) Synthesis of CRISPR/Cas9 functional modules. Cas9 mRNA and gRNA can be synthesized through in vitro transcription. Alternatively, Cas9 protein and customized gRNAs are commercially available. (A colour version of this figure is available online at: https://academic.oup.com/bfg) Transcription activator-like effector-based nucleases Similar to ZFNs, TALENs contain FokI nuclease domains but use naturally existing transcription activator-like effectors (TALEs) in the plant pathogenic bacteria Xanthomonas spp [43] for DNA recognition (Figure 2B). The DNA-binding ability of TALEs is mediated by the central domains consisting of multiple repeat units. Each unit contains 31–33 amino acids shared among all units and two variable amino acids called ‘repeat variable di-residues’ (RVDs) for determining the binding specificity [44, 45]. More than 20 different RVDs have been identified, among which NI, NG, HD and NN are the most commonly used ones that recognize nucleotides A, T, C and G/A, respectively [45, 46]. Following this code, an array of assembled repeats of TALEs can recognize virtually any user-defined sequence. TALENs have been used to target the sea urchin ETS gene [24], which plays a pivotal role in the specification of skeletogenic PMCs [47]. TALEN mRNAs targeting ETS caused skeletogenic defects in 12.6% of the injected embryos. Mutagenesis was confirmed at the sequence level, with 51.9% of the PCR clones from the injected embryos showing indels that disrupted the function of the ETS gene. The TALEN-mediated mutations appear to be generated between the unhatched blastula and the mesenchyme blastula stage, a few hours later than the mutations introduced by the ZFNs. However, a significantly smaller amount of the TALEN mRNA compared with that of ZFNs was used in these experiments. Thus, studies with better controlled conditions remain to be conducted to compare the efficiency and effective timing of ZFN- and TALEN-mediated genome editing. Clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein 9 Unlike the modular protein-based DNA recognition signature of ZFNs and TALENs, the CRISPR/Cas systems use short guide RNAs (gRNAs) to recognize target DNA (Figure 2C). The CRISPR/Cas systems were discovered as an immune defense mechanism in bacteria that recognizes and degrades invading DNA [48, 49]. Three major types (I, II and III) and many subtypes of these systems exist in nature, among which the type II CRISPR/Cas9 derived from Streptococcus pyogenes is most commonly used. The CRISPR/Cas9 system is composed of a Cas9 endonuclease and either a CRISPR RNA (crRNA)/trans-activating crRNA (tracrRNA) pair or a chimeric gRNA [50, 51]. The crRNA and the 5′ end of the gRNA contain a 20-nucleotide guide sequence responsible for the recognition of target sites, while the tracrRNA and the 3′ end of the gRNA interact with the Cas9 endonuclease. When the Cas9-RNA complexes are introduced into cells, they first scan the genome for a protospacer-adjacent motif (PAM), which is NGG for Cas9, and unwind the adjacent DNA for the formation of a heteroduplex composed of the gRNA and its targeted DNA. The endonuclease activity of Cas9 is then triggered and creates a DSB. Because of its RNA-based DNA recognition mechanism, this system provides a simple and scalable approach that empowers researchers to explore the functions of genomic sequences at the systems level. CRISPR/Cas9 technology has recently been successfully used in the sea urchin S. purpuratus. The first study designed six gRNAs targeting the open reading frame of the nodal gene, an upstream factor of the ectoderm GRNs [25]. On injection of sea urchin zygotes with in vitro-synthesized Cas9 mRNA and one of the designed gRNAs, five of the gRNAs were able to induce the expected phenotype in 60–80% of the injected embryos. Analysis of the mutation rates from individual injected embryos revealed that 67–100% of the sequenced clones contained indels at the target sites, indicating that biallelic genomic modifications were introduced. The high efficiency of the CRISPR/Cas9 system is also observed in two recent studies in which multiple gRNAs were combined to target the polyketide synthase 1 (PKS1), the glial cell missing (GCM) or the nanos2 gene [27, 28]. PKS1 encodes an enzyme that is essential for pigment synthesis in sea urchin embryos and is transcriptionally activated by the transcription factor GCM [52]. The effects of CRISPR/Cas9 on both PKS1 and GCM were highly penetrant and generated albino embryos efficiently (>95% albinism in the injected embryos) [28]. Nanos2 encodes a RNA-binding protein and is specifically expressed in the translationally quiescent primordial germ cells of the sea urchin embryo [53]. CRISPR/Cas9 targeting to the nanos2 gene resulted in high-efficiency mutations and an increase in translational activity specifically in the primordial germ cells (PGCs) [27]. The high efficiency of the CRISPR/Cas9 system may be attributed to its activity in introducing mutations during early cleavage stages. Comparison of the three genome editing tools Choosing a better genome editing approach is essential for successful downstream applications. Here, we compare ZFNs, TALENs and CRISPR/Cas9 for their synthesis strategies and editing efficiencies. Two modules including DNA-targeting element and DNA-cleaving nuclease are required for all three editing tools. Screening for functional ZFNs involves multiple steps, including two rounds of selection, fusion of zinc-finger arrays with a FokI nuclease domain, and verification using single-strand annealing (SSA) assays [41, 54] (Figure 3A). For TALENs, the assembly of TALE arrays can be technically challenging because of the high sequence similarity among the repeat units. Various methods have been developed to overcome this difficulty, among which the most commonly used approach is the Golden Gate assembly, a strategy based on type IIS restriction enzymes that cleave DNA outside their binding sites [55]. It thus removes the recognition sequences from the assembly to create seamless arrays of orderly assembled repeats (Figure 3B). Similar to ZFNs, TALE repeats need to be fused with a FokI domain and tested for efficacy. For the CRISPR/Cas9 system, Cas9 and gRNAs are often synthesized separately. Cas9 mRNA can be transcribed in vitro; alternatively, commercially available Cas9 protein can be used directly. gRNAs can also be transcribed in vitro or customized commercially. Based on the published results in sea urchins, the genome editing efficiency of the CRISPR/Cas9 system is significantly higher than those of ZFNs and TALENs (Table 1). The editing events of CRISPR/Cas9 were also detected earlier than those of ZFNs and TALENs. Considering the efficiency and timing of mutagenesis, CRISPR/Cas9 is a better tool in sea urchins. However, this comparison is drawn from limited studies; thus, a comparison of parallel experiments testing the efficiency of the three methods would be more informative. Table 1. Comparison of ZFNs, TALENs and CRISPR/Cas9 in sea urchin embryos Genome-editing approach . ZFNs . TALENs . CRISPR/Cas9 . Target gene HesC ETS nodal PKS1, GCM nanos2 Concentration 13 pg/embryo (each) 1 pg/embryo (each) 750 ng/µl Cas9, 150 ng/µl gRNAa 500 ng/µl Cas9, 400 ng/µl of each gRNA 500 ng/µl Cas9, 400 ng/µl of each gRNA Species Hemicentrotus pulcherrimus Hemicentrotus pulcherrimus Strongylocentrotus purpuratus Strongylocentrotus purpuratus Strongylocentrotus purpuratus Genome-editing efficiency  Morphological impairment (% of the embryos) 9.5 12.6 60–80 >95 N.D.  Sequence change (% of the PCR clones) 44 52 67–100 N.D. 80 Onset of mutagenesis 8-cell to unhatched blastula Unhatched blastula to mesenchyme blastula 8-cell N.D. N.D. Genome-editing approach . ZFNs . TALENs . CRISPR/Cas9 . Target gene HesC ETS nodal PKS1, GCM nanos2 Concentration 13 pg/embryo (each) 1 pg/embryo (each) 750 ng/µl Cas9, 150 ng/µl gRNAa 500 ng/µl Cas9, 400 ng/µl of each gRNA 500 ng/µl Cas9, 400 ng/µl of each gRNA Species Hemicentrotus pulcherrimus Hemicentrotus pulcherrimus Strongylocentrotus purpuratus Strongylocentrotus purpuratus Strongylocentrotus purpuratus Genome-editing efficiency  Morphological impairment (% of the embryos) 9.5 12.6 60–80 >95 N.D.  Sequence change (% of the PCR clones) 44 52 67–100 N.D. 80 Onset of mutagenesis 8-cell to unhatched blastula Unhatched blastula to mesenchyme blastula 8-cell N.D. N.D. aNote. Concentration in injecting solution; about 4 pl was injected into each embryo. N.D. = No data. Table 1. Comparison of ZFNs, TALENs and CRISPR/Cas9 in sea urchin embryos Genome-editing approach . ZFNs . TALENs . CRISPR/Cas9 . Target gene HesC ETS nodal PKS1, GCM nanos2 Concentration 13 pg/embryo (each) 1 pg/embryo (each) 750 ng/µl Cas9, 150 ng/µl gRNAa 500 ng/µl Cas9, 400 ng/µl of each gRNA 500 ng/µl Cas9, 400 ng/µl of each gRNA Species Hemicentrotus pulcherrimus Hemicentrotus pulcherrimus Strongylocentrotus purpuratus Strongylocentrotus purpuratus Strongylocentrotus purpuratus Genome-editing efficiency  Morphological impairment (% of the embryos) 9.5 12.6 60–80 >95 N.D.  Sequence change (% of the PCR clones) 44 52 67–100 N.D. 80 Onset of mutagenesis 8-cell to unhatched blastula Unhatched blastula to mesenchyme blastula 8-cell N.D. N.D. Genome-editing approach . ZFNs . TALENs . CRISPR/Cas9 . Target gene HesC ETS nodal PKS1, GCM nanos2 Concentration 13 pg/embryo (each) 1 pg/embryo (each) 750 ng/µl Cas9, 150 ng/µl gRNAa 500 ng/µl Cas9, 400 ng/µl of each gRNA 500 ng/µl Cas9, 400 ng/µl of each gRNA Species Hemicentrotus pulcherrimus Hemicentrotus pulcherrimus Strongylocentrotus purpuratus Strongylocentrotus purpuratus Strongylocentrotus purpuratus Genome-editing efficiency  Morphological impairment (% of the embryos) 9.5 12.6 60–80 >95 N.D.  Sequence change (% of the PCR clones) 44 52 67–100 N.D. 80 Onset of mutagenesis 8-cell to unhatched blastula Unhatched blastula to mesenchyme blastula 8-cell N.D. N.D. aNote. Concentration in injecting solution; about 4 pl was injected into each embryo. N.D. = No data. Overall, CRISPR/Cas9 is easier to synthesize and offers potentially higher efficiency and earlier timing of mutagenesis. However, one limitation of the CRISPR/Cas9 system is that target sites need to be adjacent to a PAM sequence. The single-base recognition of TALENs offers greater design flexibility than the triplet-based ZFNs and the PAM-dependent CRISPR/Cas9. Therefore, TALENs would complement the CRISPR/Cas9 system when target sites have to be restricted to a certain region of a gene without PAM sequences. Other applications of the CRISPR/Cas9 technology The CRISPR/Cas9 system has become the most widely used genome engineering approach because of its high efficiency, easy design principles and suitability for multiplexing. In addition to disrupting gene functions, modified versions of this system have been developed for generating transgenic animals, investigating functions of noncoding genomes, live imaging of genomes, repressing or activating transcription of genes through epigenetic modification and RNA editing. Many of these applications would greatly benefit studies of sea urchin development and thus await testing and optimization in sea urchin embryos. Targeted transgenesis Currently, transgenesis in sea urchins relies on nontargeted gene transfer systems through microinjections of linearized DNA molecules that concatenate and are randomly incorporated into the genome [56, 57]. Using this method, incorporation of transgenes, such as reporter constructs, often occurs during the third or fourth cell division in a fraction of embryonic cells, resulting in mosaic patterns of reporter gene expression [58]. Other gene-transfer techniques, those mediated by I-SceI meganuclease [59, 60], Minos transposase [61] and pantropic retrovirus [62], have also been tested in sea urchin embryos. Improved transgenic efficiency (96.5%) was achieved with retroviral transduction of an exogenous reporter construct driven by a ubiquitous Otx enhancer [62]. Despite the great success in delivering exogenous DNA into sea urchin embryos, the major limitation of the aforementioned transgenic approaches is the lack of control over where the transgenic integration occurs. Targeted transgenesis, in which transgenes integrate at desired locations in the genome, can be achieved through HDR in the presence of a donor template. To date, the only published case of targeted transgenesis in sea urchins used ZFNs to insert a GFP cassette into the coding sequence of the ETS1 gene [63]. By injecting a pair of ZFNs targeting the ETS1 gene and a donor vector containing ∼1 kb homology arms flanking a GFP cassette, GFP signals were detected in the ETS1-expressing cells. The efficiency was increased to 15% when the activity of DNA ligase IV, a functional element of the NHEJ pathway, was repressed by a dominant-negative construct. The CRISPR/Cas9 system has been applied in other model organisms for precise gene insertions. Three forms of donor vectors are commonly used: single-stranded oligodeoxynucleotides (ssODNs) with short homology arms (50–80 bp), double-stranded DNA with long homology arms (∼800 bp) and double-stranded DNA with short homologous sequences (10–40 bp) flanked by two Cas9 cleavage sites. A double-stranded DNA donor can integrate much longer exogenous DNA sequences such as reporter genes. The precise in-frame insertion of a fluorescence reporter in developmental genes can be used for lineage tracing, deep-tissue imaging and cell sorting (Figure 4A). The HDR efficiency using ssODNs is generally higher; however, because of its length restriction, its application is limited to shorter sequence modifications, such as introducing point mutations or short functional sites (enzyme recognition sites or the loxP site) (Figure 4A). We expect that these approaches will soon be applied in the sea urchin system to improve the efficiency of targeted transgenesis. Figure 4: Open in new tabDownload slide Other applications of the CRISPR/Cas9 system. (A) The application of CRISPR/Cas9 for gene knock-in (left) or the integration of short fragments (right). (B) CRISPR/Cas9 targets regulatory sequences for studying cis-regulation of genes. Two gRNAs are shown to bind to each side of the targeted enhancer. This approach can also be applied to delete other regulatory elements, such as promoters, repressors and insulators. (C) Catalytically dCas9 is fused with a repressor (KRAB) or an activator (VP64 or P300) to repress (CRISPRi) or activate (CRISPRa) gene transcription, respectively. (D) The application of CRISPR/Cas9 for RNA targeting. The RNA-targeting capability of Cas9 relies on a PAMmer. Cas9 triggers the site-specific cleavage of RNA, leading to its degradation. (A colour version of this figure is available online at: https://academic.oup.com/bfg) Figure 4: Open in new tabDownload slide Other applications of the CRISPR/Cas9 system. (A) The application of CRISPR/Cas9 for gene knock-in (left) or the integration of short fragments (right). (B) CRISPR/Cas9 targets regulatory sequences for studying cis-regulation of genes. Two gRNAs are shown to bind to each side of the targeted enhancer. This approach can also be applied to delete other regulatory elements, such as promoters, repressors and insulators. (C) Catalytically dCas9 is fused with a repressor (KRAB) or an activator (VP64 or P300) to repress (CRISPRi) or activate (CRISPRa) gene transcription, respectively. (D) The application of CRISPR/Cas9 for RNA targeting. The RNA-targeting capability of Cas9 relies on a PAMmer. Cas9 triggers the site-specific cleavage of RNA, leading to its degradation. (A colour version of this figure is available online at: https://academic.oup.com/bfg) Cis-regulatory analysis at genomic loci The construction of GRN models often requires knowledge of direct regulatory interactions. Current methods used to study and identify these cis-regulatory interactions in sea urchins rely on reporter systems [64], in which genomic sequences are amplified and measured for their ability to drive reporter expression outside of their genomic context. The CRISPR/Cas9 system has been used to study the functions of regulatory sequences at endogenous genomic loci [65, 66] (Figure 4B). To delete the entire sequence of a module, two gRNAs targeting both ends of the module need to be delivered simultaneously with Cas9. DSBs at both target sites trigger NHEJ-mediated DNA repair and lead to the genomic deletion of the targeted module. CRISPR/Cas9 can also be used to disrupt putative binding sites to infer direct upstream regulators. This application also offers the opportunity to study chromosomal architectures and how repressors and insulators regulate gene transcription, which would not be possible with reporter assays. Deletion of specific regulatory modules in the sea urchin genome is potentially feasible. However, because regulatory sequences are usually located in noncoding regions, where polymorphism is generally high, re-sequencing the genomic sequences around potential gRNA target sites may be necessary. Additionally, because the deletion of a DNA fragment requires two gRNAs acting at the same time, choosing two gRNAs with similar efficiencies would increase the chance of fragment deletion instead of creating short indels resulting from actions of a single gRNA. Transcriptional modulation by CRISPR/dead Cas9 CRISPR interference (CRISPRi) uses a catalytically dead Cas9 (dCas9) to block transcription initiation or elongation [67]. The repressive function of CRISPRi can be further enhanced by fusing dCas9 to the repressive Krüppel-associated box (KRAB) domain, which promotes epigenetic silencing [68] (Figure 4C). Similarly, by tethering to transcription activation domains such as VP16/VP64 or P300, dCas9 can be converted into a synthetic transcriptional activator [69, 70] (Figure 4C). This dCas9-mediated activation (CRISPRa) upregulates the expression of genes from their genomic loci, offering an overexpression tool for studying gain-of-function phenotypes. The position of gRNA targets is critical for this application. A high-throughput assay, in which thousands of gRNAs were designed to target regions around transcription start sites (+1) of 49 genes, revealed that the highest levels of repression and activation were achieved by targeting the −50 to 300 bp region and the −400 to −50 bp region, respectively [71]. CRISPR/dCas9 can be combined with other tools to modulate gene expression in a spatially and temporally specific manner. For example, light-activated dCas9-effectors are based on optogenetics, which uses light-induced heterodimerization. By fusing blue-light-sensing system cryptochrome (CRY) and CIB domains to dCas9 and the transcriptional activators VP64 or p65AD, recent studies demonstrated the activation of endogenous genes on illuminating cells with blue light [72, 73]. An inducible system based on rapamycin-dependent dimerization has also been developed for activating endogenous genes [74]. These studies demonstrated that targeted gene regulation could be spatially and temporally controlled in a reversible manner, which is useful for understanding dynamic gene networks. CRISPR/Cas9 for RNA recognition Native Cas9 acts exclusively on DNA substrates because of its requirement for the PAM sequence. It has been shown that when the PAM is provided in trans as an additional DNA oligonucleotide, Cas9 is able to target single-stranded RNA (ssRNA) [75] (Figure 4D). Similar to PAM-mediated DNA cleavage, this PAM-presenting oligonucleotide (PAMmer) is able to trigger the site-specific cleavage of ssRNA. Interestingly, this system can tolerate mismatch with ssRNA at the NGG site, as long as additional nucleotides upstream of the NGG are present in the PAMmers to direct the binding. The length of two to eight additional nucleotides has shown to be optimal for specific targeting without compromising the binding affinity and cleavage efficiency [75]. This tolerance of mismatch at the NGG site allows researchers to target any ssRNA without having the constraint of searching for NGG-adjacent gRNA targets. Other RNA-binding CRISPR systems include the CRISPR/Cas type III-B Cmr complex [76] and atypical Cas9 from Francisella novicida [77]. Recently, a Class 2 type VI-A CRISPR/Cas effector C2c2 from Leptotrichia shahii has been demonstrated as the first example of a CRISPR/Cas9 system that exclusively targets RNA [78]. The ability of Cas9 to target RNA allows for the selective alteration of RNA without changing the genomic sequence. In addition to RNA degradation, several potential applications have been discussed for RNA-targeted Cas9 in live cells [79]. For example, by replacing Cas9 with dCas9 fused to a fluorescent protein or biotin, this system allows the visualization of RNA trafficking in living cells and the isolation of the endogenous RNA population [75, 80]. Conclusion Functional perturbation is essential to unfold developmental processes into logic maps of regulatory network models. Since the publication of the sea urchin genome in 2006, several GRNs controlling various developmental processes have been established, based mostly on perturbations using regular MOs injected into zygotes. The new technologies described in this review, including vivo-MOs and targeted genome editing, provide flexibility in the temporal and spatial control of functional perturbations in sea urchins. We especially look forward to combine previously established knowledge with the fast-developing CRISPR tools. For example, previously identified tissue-specific enhancers from sea urchins can be used to activate tissue-specific expression of Cas9, resulting in spatial control of genome editing. A photoactivatable Cas9 system can also be used to control the timing of genome editing [81]. New CRISPR endonucleases such as Cpf1 have been discovered and tested in several model organisms [82]. These new CRISPR effectors have provided more possibilities in choosing target sites. We envision that with the development of new technologies, studies on sea urchin development will come into an exciting era. Supplementary data Supplementary data are available online at https://academic.oup.com/bfg. Key Points Effective perturbation of gene functions by using antisense MOs is the most widely used approach for establishing sea urchin GRNs. The modification of morpholinos into membrane permeable vivo-MOs allows gene knockdown at later developmental stages. Several genome editing tools have been successfully applied in sea urchins for gene knockout. Various applications of the CRISPR/Cas9 system allow the functional perturbation of gene functions in more versatile ways. Funding Y.-H.S. is supported by the Ministry of Science and Technology, Taiwan (grant numbers 103-2311-B-001 -030 -MY3, 104-2627-B-001-001 and 105-2321-B-001-035). Miao Cui is a postdoctoral fellow at the Department of Neurological Surgery, University of California, San Francisco. Her research interest involves understanding the developmental roles of noncoding genome. She conducted her PhD research in the laboratory of Dr Eric Davidson at California Institute of Technology on sea urchin developmental gene networks. Che-Yi Lin is a postdoctoral fellow at the Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan. His current research focuses on the genome editing applications and single-cell RNA-seq analyses. Yi-Hsien Su is an associate research fellow in the Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan. Her research group is interested in the evolution of developmental mechanisms controlling animal body plans. References 1 Ernst SG. A century of sea urchin development . Am Zool 1997 ; 37 : 250 – 9 . Google Scholar Crossref Search ADS WorldCat 2 Davidson EH. Network design principles from the sea urchin embryo . Curr Opin Genet Dev 2009 ; 19 : 535 – 40 . Google Scholar Crossref Search ADS PubMed WorldCat 3 McClay DR. Evolutionary crossroads in developmental biology: sea urchins . Development 2011 ; 138 : 2639 – 48 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Duboc V , Rottinger E, Besnardeau L, et al. Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo . Dev Cell 2004 ; 6 : 397 – 410 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Luo YJ , Su YH. Opposing Nodal and BMP signals regulate left-right asymmetry in the sea urchin larva . PLoS Biol 2012 ; 10 : e1001402. Google Scholar Crossref Search ADS PubMed WorldCat 6 Materna SC , Davidson EH. A comprehensive analysis of Delta signaling in pre-gastrular sea urchin embryos . Dev Biol 2012 ; 364 : 77 – 87 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Andrikou C , Pai CY, Su YH, et al. Logics and properties of a genetic regulatory program that drives embryonic muscle development in an echinoderm . Elife 2015 ; 4 : e07343 . Google Scholar Crossref Search ADS WorldCat 8 Cui M , Siriwon N, Li E, et al. Specific functions of the Wnt signaling system in gene regulatory networks throughout the early sea urchin embryo . Proc Natl Acad Sci USA 2014 ; 111 : E5029 – 38 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Walton KD , Warner J, Hertzler PH, et al. Hedgehog signaling patterns mesoderm in the sea urchin . Dev Biol 2009 ; 331 : 26 – 37 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Materna SC , Swartz SZ, Smith J. Notch and Nodal control forkhead factor expression in the specification of multipotent progenitors in sea urchin . Development 2013 ; 140 : 1796 – 806 . Google Scholar Crossref Search ADS PubMed WorldCat 11 Oliveri P , Carrick DM, Davidson EH. A regulatory gene network that directs micromere specification in the sea urchin embryo . Dev Biol 2002 ; 246 : 209 – 28 . Google Scholar Crossref Search ADS PubMed WorldCat 12 Range RC , Wei Z. An anterior signaling center patterns and sizes the anterior neuroectoderm of the sea urchin embryo . Development 2016 ; 143 : 1523 – 33 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Ransick A , Davidson EH. cis-regulatory processing of Notch signaling input to the sea urchin glial cells missing gene during mesoderm specification . Dev Biol 2006 ; 297 : 587 – 602 . Google Scholar Crossref Search ADS PubMed WorldCat 14 Rottinger E , Saudemont A, Duboc V, et al. FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis and regulate gastrulation during sea urchin development . Development 2008 ; 135 : 353 – 65 . Google Scholar Crossref Search ADS PubMed WorldCat 15 Saunders LR , McClay DR. Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition . Development 2014 ; 141 : 1503 – 13 . Google Scholar Crossref Search ADS PubMed WorldCat 16 Yaguchi S , Yaguchi J, Burke RD. Specification of ectoderm restricts the size of the animal plate and patterns neurogenesis in sea urchin embryos . Development 2006 ; 133 : 2337 – 46 . Google Scholar Crossref Search ADS PubMed WorldCat 17 Annunziata R , Arnone MI. A dynamic regulatory network explains ParaHox gene control of gut patterning in the sea urchin . Development 2014 ; 141 : 2462 – 72 . Google Scholar Crossref Search ADS PubMed WorldCat 18 Saudemont A , Haillot E, Mekpoh F, et al. Ancestral regulatory circuits governing ectoderm patterning downstream of Nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm . PLoS Genet 2010 ; 6 : e1001259 . Google Scholar Crossref Search ADS PubMed WorldCat 19 Su YH , Li E, Geiss GK, et al. A perturbation model of the gene regulatory network for oral and aboral ectoderm specification in the sea urchin embryo . Dev Biol 2009 ; 329 : 410 – 21 . Google Scholar Crossref Search ADS PubMed WorldCat 20 Martik ML , McClay DR. Deployment of a retinal determination gene network drives directed cell migration in the sea urchin embryo . Elife 2015 ; 4 : e08827 . Google Scholar Crossref Search ADS PubMed WorldCat 21 Angerer LM , Angerer RC. Disruption of gene function using antisense morpholinos . Methods Cell Biol 2004 ; 74 : 699 – 711 . Google Scholar Crossref Search ADS PubMed WorldCat 22 Morcos PA , Li Y, Jiang S. Vivo-morpholinos: a non-peptide transporter delivers morpholinos into a wide array of mouse tissues . Biotechniques 2008 ; 45 : 613 – 14 , 616, 618 passim. Google Scholar Crossref Search ADS PubMed WorldCat 23 Heyland A , Hodin J, Bishop C. Manipulation of developing juvenile structures in purple sea urchins (Strongylocentrotus purpuratus) by morpholino injection into late stage larvae . PLoS One 2014 ; 9 : e113866. Google Scholar Crossref Search ADS PubMed WorldCat 24 Hosoi S , Sakuma T, Sakamoto N, et al. Targeted mutagenesis in sea urchin embryos using TALENs . Dev Growth Differ 2014 ; 56 : 92 – 7 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Lin CY , Su YH. Genome editing in sea urchin embryos by using a CRISPR/Cas9 system . Dev Biol 2016 ; 409 : 420 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 26 Ochiai H , Fujita K, Suzuki K, et al. Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases . Genes Cells 2010 ; 15 : 875 – 85 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 27 Oulhen N , Swartz SZ, Laird J, et al. Transient translational quiescence in primordial germ cells . Development 2017 , 144: 1201 – 10 . Google Scholar Crossref Search ADS PubMed WorldCat 28 Oulhen N , Wessel GM. Albinism as a visual, in vivo guide for CRISPR/Cas9 functionality in the sea urchin embryo . Mol Reprod Dev 2016 ; 83 : 1046 – 7 . Google Scholar Crossref Search ADS PubMed WorldCat 29 Lepage T , Gache C. Expression of exogenous mRNAs to study gene function in the sea urchin embryo . Methods Cell Biol 2004 ; 74 : 677 – 97 . Google Scholar Crossref Search ADS PubMed WorldCat 30 Cheers MS , Ettensohn CA. P16 is an essential regulator of skeletogenesis in the sea urchin embryo . Dev Biol 2005 ; 283 : 384 – 96 . Google Scholar Crossref Search ADS PubMed WorldCat 31 Adomako-Ankomah A , Ettensohn CA. P58-A and P58-B: novel proteins that mediate skeletogenesis in the sea urchin embryo . Dev Biol 2011 ; 353 : 81 – 93 . Google Scholar Crossref Search ADS PubMed WorldCat 32 Summerton JE. Endo-Porter: a novel reagent for safe, effective delivery of substances into cells . Ann N Y Acad Sci 2005 ; 1058 : 62 – 75 . Google Scholar Crossref Search ADS PubMed WorldCat 33 Tallafuss A , Gibson D, Morcos P, et al. Turning gene function ON and OFF using sense and antisense photo-morpholinos in zebrafish . Development 2012 ; 139 : 1691 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 34 Liu Q , Segal DJ, Ghiara JB, et al. Design of polydactyl zinc-finger proteins for unique addressing within complex genomes . Proc Natl Acad Sci USA 1997 ; 94 : 5525 – 30 . Google Scholar Crossref Search ADS PubMed WorldCat 35 Beerli RR , Barbas CF III. Engineering polydactyl zinc-finger transcription factors . Nat Biotechnol 2002 ; 20 : 135 – 41 . Google Scholar Crossref Search ADS PubMed WorldCat 36 Bhakta MS , Henry IM, Ousterout DG, et al. Highly active zinc-finger nucleases by extended modular assembly . Genome Res 2013 ; 23 : 530 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 37 Kim S , Lee MJ, Kim H, et al. Preassembled zinc-finger arrays for rapid construction of ZFNs . Nat Methods 2011 ; 8 : 7 . Google Scholar Crossref Search ADS PubMed WorldCat 38 Maeder ML , Thibodeau-Beganny S, Osiak A, et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification . Mol Cell 2008 ; 31 : 294 – 301 . Google Scholar Crossref Search ADS PubMed WorldCat 39 Meng X , Brodsky MH, Wolfe SA. A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors . Nat Biotechnol 2005 ; 23 : 988 – 94 . Google Scholar Crossref Search ADS PubMed WorldCat 40 Pabo CO , Peisach E, Grant RA. Design and selection of novel Cys2His2 zinc finger proteins . Annu Rev Biochem 2001 ; 70 : 313 – 40 . Google Scholar Crossref Search ADS PubMed WorldCat 41 Meng X , Noyes MB, Zhu LJ, et al. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases . Nat Biotechnol 2008 ; 26 : 695 – 701 . Google Scholar Crossref Search ADS PubMed WorldCat 42 Revilla-i-Domingo R , Oliveri P, Davidson EH. A missing link in the sea urchin embryo gene regulatory network: hesC and the double-negative specification of micromeres . Proc Natl Acad Sci USA 2007 ; 104 : 12383 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 43 Boch J , Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function . Annu Rev Phytopathol 2010 ; 48 : 419 – 36 . Google Scholar Crossref Search ADS PubMed WorldCat 44 Boch J , Scholze H, Schornack S, et al. Breaking the code of DNA binding specificity of TAL-type III effectors . Science 2009 ; 326 : 1509 – 12 . Google Scholar Crossref Search ADS PubMed WorldCat 45 Moscou MJ , Bogdanove AJ. A simple cipher governs DNA recognition by TAL effectors . Science 2009 ; 326 : 1501 . Google Scholar Crossref Search ADS PubMed WorldCat 46 Juillerat A , Pessereau C, Dubois G, et al. Optimized tuning of TALEN specificity using non-conventional RVDs . Sci Rep 2015 ; 5 : 8150. Google Scholar Crossref Search ADS PubMed WorldCat 47 Kurokawa D , Kitajima T, Mitsunaga-Nakatsubo K, et al. HpEts, an ets-related transcription factor implicated in primary mesenchyme cell differentiation in the sea urchin embryo . Mech Dev 1999 ; 80 : 41 – 52 . Google Scholar Crossref Search ADS PubMed WorldCat 48 Barrangou R , Marraffini LA. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity . Mol Cell 2014 ; 54 : 234 – 44 . Google Scholar Crossref Search ADS PubMed WorldCat 49 Deveau H , Garneau JE, Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions . Annu Rev Microbiol 2010 ; 64 : 475 – 93 . Google Scholar Crossref Search ADS PubMed WorldCat 50 Cong L , Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems . Science 2013 ; 339 : 819 – 23 . Google Scholar Crossref Search ADS PubMed WorldCat 51 Mali P , Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9 . Science 2013 ; 339 : 823 – 6 . Google Scholar Crossref Search ADS PubMed WorldCat 52 Calestani C , Rogers DJ. Cis-regulatory analysis of the sea urchin pigment cell gene polyketide synthase . Dev Biol 2010 ; 340 : 249 – 55 . Google Scholar Crossref Search ADS PubMed WorldCat 53 Juliano CE , Yajima M, Wessel GM. Nanos functions to maintain the fate of the small micromere lineage in the sea urchin embryo . Dev Biol 2010 ; 337 : 220 – 32 . Google Scholar Crossref Search ADS PubMed WorldCat 54 Szczepek M , Brondani V, Buchel J, et al. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases . Nat Biotechnol 2007 ; 25 : 786 – 93 . Google Scholar Crossref Search ADS PubMed WorldCat 55 Cermak T , Doyle EL, Christian M, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting . Nucleic Acids Res 2011 ; 39 : e82. Google Scholar Crossref Search ADS PubMed WorldCat 56 Franks RR , Hough-Evans BR, Britten RJ, et al. Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA . Development 1988 ; 102 : 287 – 99 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 57 McMahon AP , Flytzanis CN, Hough-Evans BR, et al. Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis . Dev Biol 1985 ; 108 : 420 – 30 . Google Scholar Crossref Search ADS PubMed WorldCat 58 Hough-Evans BR , Britten RJ, Davidson EH. Mosaic incorporation and regulated expression of an exogenous gene in the sea urchin embryo . Dev Biol 1988 ; 129 : 198 – 208 . Google Scholar Crossref Search ADS PubMed WorldCat 59 Akasaka K , Nishimura A, Takata K, et al. Upstream element of the sea urchin arylsulfatase gene serves as an insulator . Cell Mol Biol 1999 ; 45 : 555 – 65 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 60 Ochiai H , Sakamoto N, Suzuki K, et al. The Ars insulator facilitates I-SceI meganuclease-mediated transgenesis in the sea urchin embryo . Dev Dyn 2008 ; 237 : 2475 – 82 . Google Scholar Crossref Search ADS PubMed WorldCat 61 Sasakura Y , Yaguchi J, Yaguchi S, et al. Excision and transposition activity of Tc1/mariner superfamily transposons in sea urchin embryos . Zoolog Sci 2010 ; 27 : 256 – 62 . Google Scholar Crossref Search ADS PubMed WorldCat 62 Core AB , Reyna AE, Conaway EA, et al. Pantropic retroviruses as a transduction tool for sea urchin embryos . Proc Natl Acad Sci USA 2012 ; 109 : 5334 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 63 Ochiai H , Sakamoto N, Fujita K, et al. Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos . Proc Natl Acad Sci USA 2012 ; 109 : 10915 – 20 . Google Scholar Crossref Search ADS PubMed WorldCat 64 Nam J , Dong P, Tarpine R, et al. Functional cis-regulatory genomics for systems biology . Proc Natl Acad Sci USA 2010 ; 107 : 3930 – 5 . Google Scholar Crossref Search ADS PubMed WorldCat 65 Canver MC , Smith EC, Sher F, et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis . Nature 2015 ; 527 : 192 – 7 . Google Scholar Crossref Search ADS PubMed WorldCat 66 Korkmaz G , Lopes R, Ugalde AP, et al. Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9 . Nat Biotechnol 2016 ; 34 : 192 – 8 . Google Scholar Crossref Search ADS PubMed WorldCat 67 Larson MH , Gilbert LA, Wang X, et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression . Nat Protoc 2013 ; 8 : 2180 – 96 . Google Scholar Crossref Search ADS PubMed WorldCat 68 Gilbert LA , Larson MH, Morsut L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes . Cell 2013 ; 154 : 442 – 51 . Google Scholar Crossref Search ADS PubMed WorldCat 69 Hilton IB , D'Ippolito AM, Vockley CM, et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers . Nat Biotechnol 2015 ; 33 : 510 – 17 . Google Scholar Crossref Search ADS PubMed WorldCat 70 Maeder ML , Linder SJ, Cascio VM, et al. CRISPR RNA-guided activation of endogenous human genes . Nat Methods 2013 ; 10 : 977 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 71 Gilbert LA , Horlbeck MA, Adamson B, et al. Genome-scale CRISPR-mediated control of gene repression and activation . Cell 2014 ; 159 : 647 – 61 . Google Scholar Crossref Search ADS PubMed WorldCat 72 Nihongaki Y , Yamamoto S, Kawano F, et al. CRISPR-Cas9-based photoactivatable transcription system . Chem Biol 2015 ; 22 : 169 – 74 . Google Scholar Crossref Search ADS PubMed WorldCat 73 Polstein LR , Gersbach CA. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation . Nat Chem Biol 2015 ; 11 : 198 – 200 . Google Scholar Crossref Search ADS PubMed WorldCat 74 Zetsche B , Volz SE, Zhang F. A split-Cas9 architecture for inducible genome editing and transcription modulation . Nat Biotechnol 2015 ; 33 : 139 – 42 . Google Scholar Crossref Search ADS PubMed WorldCat 75 O'Connell MR , Oakes BL, Sternberg SH, et al. Programmable RNA recognition and cleavage by CRISPR/Cas9 . Nature 2014 ; 516 : 263 – 6 . Google Scholar Crossref Search ADS PubMed WorldCat 76 Hale CR , Zhao P, Olson S, et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex . Cell 2009 ; 139 : 945 – 56 . Google Scholar Crossref Search ADS PubMed WorldCat 77 Sampson TR , Saroj SD, Llewellyn AC, et al. A CRISPR/Cas system mediates bacterial innate immune evasion and virulence . Nature 2013 ; 497 : 254 – 7 . Google Scholar Crossref Search ADS PubMed WorldCat 78 Abudayyeh OO , Gootenberg JS, Konermann S, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector . Science 2016 ; 353 : aaf5573. Google Scholar Crossref Search ADS PubMed WorldCat 79 Nelles DA , Fang MY, Aigner S, et al. Applications of Cas9 as an RNA-programmed RNA-binding protein . Bioessays 2015 ; 37 : 732 – 9 . Google Scholar Crossref Search ADS PubMed WorldCat 80 Nelles DA , Fang MY, O'Connell MR, et al. Programmable RNA tracking in live cells with CRISPR/Cas9 . Cell 2016 ; 165 : 488 – 96 . Google Scholar Crossref Search ADS PubMed WorldCat 81 Nihongaki Y , Kawano F, Nakajima T, et al. Photoactivatable CRISPR-Cas9 for optogenetic genome editing . Nat Biotechnol 2015 ; 33 : 755 – 60 . Google Scholar Crossref Search ADS PubMed WorldCat 82 Zetsche B , Gootenberg JS, Abudayyeh OO, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system . Cell 2015 ; 163 : 759 – 71 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes * " These authors contributed equally to this work © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com
TI - Recent advances in functional perturbation and genome editing techniques in studying sea urchin development
JF - Briefings in Functional Genomics
DO - 10.1093/bfgp/elx011
DA - 2017-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/recent-advances-in-functional-perturbation-and-genome-editing-Zp6v0AJ4Ap
SP - 309
VL - 16
IS - 5
DP - DeepDyve
ER -