Nucleotide base editors in plants have been limited to conversion of cytosine to thymine. Here, we describe a new plant adenine base editor based on an evolved tRNA adenosine deaminase fused to the nickase CRISPR/Cas9, enabling A� TtoG� C conversion at frequencies up to 7.5% in protoplasts and 59.1% in regenerated rice and wheat plants. An endogenous gene is also successfully modified through introducing a gain-of-function point mutation to directly produce an herbicide-tolerant rice plant. With this new adenine base editing system, it is now possible to precisely edit all base pairs, thus expanding the toolset for precise editing in plants. Keywords: Cas9-adenosine deaminase, sgRNA forms, Rice, Wheat, Herbicide resistance Background G� C conversions when directed by single guide RNAs The CRISPR (clustered regularly interspaced short palin- (sgRNAs) to genomic targets in human cells . dromic repeat) system has been used to edit a variety of In this report, we adapted this method and optimized plant species . CRISPR/Cas9 and CRISPR/Cpf1 typically an ABE for application in plant systems, demonstrating produce double strand breaks (DSBs) that result in mutant its high efficiency in creating targeted point mutations at plants with either gene knock-outs (via non-homologous multiple endogenous loci in rice and wheat. end joining (NHEJ)) or gene replacements and insertions (via homology-directed repair (HDR)) [2, 3]. Base editing is Results a unique genome editing system that creates precise and We used ABE7.10, a fusion of an adenosine deaminase highly predictable nucleotide substitutions at genomic tar- (ecTadA-ecTadA*) with nCas9 (D10A), which base edits gets without requiring DSBs, or donor DNA templates, or A� Tto G� C accurately in human cells . To develop an depending on NHEJ and HDR . Base editing is more efficient ABE for plant cells, we constructed seven ABE efficient than HDR-mediated base pair substitution, and fusion proteins. The seven proteins, named PABE-1 to produces fewer undesirable mutations in the target locus PABE-7, varied in the position of the adenosine deaminase . The most commonly used base editing systems, such as and the number and locations of nuclear localization se- BE3 , BE4 , Targeted-AID , and dCpf1-BE , use quences (NLSs; Fig. 1a; Additional file 1: Sequences). All Cas9 or Cpf1 variants to recruit cytidine deaminases that the PABE constructs were codon-optimized for cereal exploit DNA mismatch repair pathways and generate spe- plants, and placed under control of the maize Ubiquitin-1 cific C to T substitutions. This base-editing technology has promoter (Ubi-1). already been used in a wide variety of cell lines and organ- Editing efficiencies of the PABE constructs were first isms [4, 5]. Recently, adenine base editors (ABE), developed tested using a green fluorescent protein (GFP) reporter by fusing an evolved tRNA adenosine deaminase with that contained a mutation within the expression cassette SpCas9 nickase (D10A), were shown to generate A� Tto converting the Gln-69 codon (CAG) for GFP into a stop codon (TAG) (Fig. 1b). This mutated gene, termed mGFP, * Correspondence: firstname.lastname@example.org Equal contributors produces active GFP when the stop codon is corrected by State Key Laboratory of Plant Cell and Chromosome Engineering, Center for a T to C single nucleotide substitution (TAG to CAG), Genome Editing, Institute of Genetics and Developmental Biology, Chinese thus allowing mutagenesis efficiency to be measured as Academy of Sciences, Beijing, China Full list of author information is available at the end of the article the frequency of GFP-expressing cells (Fig. 1b). We © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Genome Biology (2018) 19:59 Page 2 of 9 Fig. 1 Comparison of A� Tto G� C base-editing efficiency in rice protoplasts using seven PABE constructs. a The seven plant adenine base editing (PABE) constructs. b Diagram of the GFP reporter system for comparing the activities of the seven PABE constructs in rice protoplasts. The TAG stop codon (whose conversion to CAG restores GFP protein production) and CAG triplets are shown in the red box. c Plant ABE-induced conversion of mGFP to GFP in rice protoplasts by the seven PABE constructs. Seven fields of protoplasts transformed with the relevant PABE construct, sgRNA-mGFP and Ubi-mGFP vectors. Ubi-GFP and Ubi-mGFP served as controls. Scale bars, 150 μm. d The frequencies (percentage) of A to G conversion in the target region of the mGFP coding sequence were measured by flow cytometry (FCM) on three independent biological replicates (n = 3). All values represent means ± standard error of the mean (s.e.m.). **P < 0.01. e Frequencies of targeted single A to G conversion in reads of the 16 target sites by PABE-2 and PABE-7 in rice protoplasts. An untreated protoplast sample was used as control. Each frequency (mean ± s.e.m.) was calculated using the data from three independent biological replicates (n =3) Li et al. Genome Biology (2018) 19:59 Page 3 of 9 designed an sgRNA-mGFP with the desired T at position 6 showed the highest base editing efficiency in a large ma- (T ) of the protospacer, counting from the distal end to the jority of the tests ranging from 0.1–7.5% in both rice and protospacer-adjacent motif (PAM), based on the ABE7.10 wheat (Fig. 2a). The average efficiency of esgRNA for the deamination window in human cells (Fig. 1b; 13 target sites was about twofold higher than that of the Additional file 2: Table S1). Each PABE construct was native sgRNA, and threefold higher than that of the co-transfected with sgRNA-mGFP and Ubi-mGFP into rice tRNA-sgRNA (Fig. 2b), which is consistent with the protoplasts by PEG-mediated transformation . observation that esgRNA increases the stability and pro- At 24 h post-transfection, GFP fluorescence was reliably motes complexing with the Cas9 protein . We ob- detected in cells treated with the following five of the served only A to G conversions, with no evidence of seven test constructs: PABE-1, PABE-2, PABE-3, PABE-6, undesired editing at any of the rice and wheat genomic and PABE-7 (Fig. 1c). Flow cytometry (FCM) analyses on-target loci (< 0.02%; Additional file 2: Figures S2 and showed that the percentages of fluorescent cells ranged S3), and a much lower frequency of indels (< 0.1%) than from 0.1 to 32.8% (Fig. 1d). Three copies of the NLS at the with WT Cas9 (3.3–31.6%) (Fig. 2c). To summarize, the C-terminus of nCas9 (PABE-7) gave the highest yield of PABE-7 base editing construct, together with the esgRNA, GFP-expressing cells, higher than PABE-2, which was induces A to G substitutions efficiently and with high fi- similar to the construct used in human cells  and the delity at multiple loci in rice and wheat. other PABE constructs (Fig. 1c, d). These results also We also tested the effect of spacer length of the showed that putting ecTadA-ecTadA* adenosine deami- esgRNA on base editing efficiency by targeting OsEV nase at the C-terminus of nCas9 (PABE-4 and PABE-5) and OsOD, and found that the esgRNAs with canonical renders the plant ABE system ineffective (Fig. 1c, d). 20-nucleotide spacers showed the highest conversion To further compare the editing efficiency of PABE-2 efficiency (Fig. 2d; Additional file 2: Table S3). At both and PABE-7, we targeted 16 rice endogenous genomic target sites, esgRNAs with spacer lengths ranging from sites (Fig. 1e; Additional file 2: Table S1). A to G base 14 to 19 nucleotides showed substantially decreased or editing of the respective genes in protoplasts was undetectable A to G base editing activities (< 0.9%) com- assessed by next-generation sequencing (100,000– pared with the esgRNAs with canonical 20-nucleotide 220,000 reads per locus). PABE-7 was identified to offer spacers (< 4.5%) (Fig. 2d). In addition, the WT Cas9 with modestly higher base editing efficiency, about 1.1-fold 14- to 19-nucleotide spacer lengths of esgRNAs also average increase in A� TtoG� C conversion at each site gave much lower frequencies of indels (0.3–12.6%) than over PABE-2 (Fig. 1e; Additional file 2: Table S3). Taken with 20-nucleotide esgRNA (10.8–22.4%) at these two together, these results demonstrate that the plant ABE sites (Additional file 2: Figure S4). These results suggest system can induce A to G conversions in rice, and that that the 20-nucleotide spacer of esgRNA is essential for the presence of three NLS at the C-terminus of nCas9 the plant ABE system with no tolerance for shorter maximizes editing efficiency. lengths. To identify the optimal form of sgRNA for PABE-7 ac- To demonstrate the use of PABE-7 to regenerate rice mu- tivity, various sgRNA modifications were tested over a tant plants, we targeted six rice genomic loci (OsACC-T1, broad range of endogenous loci. Previous work has OsALS-T1, OsCDC48-T3, OsDEP1-T1, OsDEP1-T2, and shown that modifications to the sgRNA sequence OsNRT1.1B-T1) (Table 1; Additional file 2:Table S1)using (F + E) (known as sgRNA , enhanced sgRNA, or esgRNA) Agrobacterium-mediated transformation (Additional file 2:  or tRNA-sgRNA expression system [13, 14] can Figure S5a). After examination of PABE-7 with esgRNA enhance CRISPR/Cas9 genome editing. We therefore (pH-PABE-7-esgRNA)-transformed lines, substitutions were compared the base editing activities of the three sgRNA identified in all six sites in the T0 seedlings (Table 1; forms (native sgRNA, esgRNA, tRNA-sgRNA) at ten Additional file 2: Figure S5). The A to G substitution efficien- and three endogenous genomic target sites in rice and cies varied from 15.8 to 59.1%, and we identified one, six, wheat, respectively (Fig. 2a; Additional file 2: Figure S1 one, and thirteen homozygous mutant lines for OsACC-T1, and Table S1). The protospacers targeting these OsDEP1-T1, OsDEP1-T2, and OsNRT1.1B-T1, respectively endogenous genes were individually cloned into the three (Table 1; Additional file 2: Figure S5). Importantly, we sgRNA structures and co-transformed with PABE-7 into noticed that the PABE-7 conversion frequency with either rice or wheat protoplasts. Wild-type Cas9 (WT esgRNA was on average 1.7-fold higher than that ob- Cas9) was used as a control to produce deletion and/or tained with the native sgRNA constructs at each site insertion mutations (indels). A to G conversion was in side-by-side experiments (Table 1), consistent with observed at all 13 target sites for each combination of the results observed with protoplasts (Fig. 2a, b). PABE-7 and sgRNA expression system, with effective edit- Among all these six target sites, the effective deamination ing frequency spanning positions 4 to 8 within the proto- window (4 to 8) was consistent with the protoplast results. spacer (Fig. 2a). Of the three sgRNA constructs, esgRNA In addition, none of the transgenic rice plants contained Li et al. Genome Biology (2018) 19:59 Page 4 of 9 Fig. 2 Analysis of PABE-7 activity on endogenous genes using different sgRNA expression constructs. a Frequencies of targeted single A to G conversions in the 13 target sites of rice and wheat genes. The native forms of sgRNA, esgRNA, and tRNA-sgRNA were used. b Summary of the A to G conversion activities of PABE-7 in a. c Frequencies of indels in the 13 target sites of rice and wheat genes. d The effect of spacer length of sgRNA on editing efficiency. A to G editing frequencies induced by the PABE-7 and esgRNAs of different length varying from 14 to 20 nucleotides were determined at protospacer positions 2–9. In a, c, and d, an untreated protoplast sample was used as control and each frequency (mean ± standard error of the mean) was calculated using the data from three independent biological replicates (n = 3). e OsACC-T1 with C2186R substitution confers resistance to herbicide. Sequence alignment comparing WT OsACC-T1 with that in the T0–13 mutant. Phenotypes of T0–13 with C2186R substitution in the regeneration medium supplemented with 0.086 ppm haloxyfop-R-methyl. Scale bars,1 cm any indels or undesired edits at the target site (Additional pathways, and mutations in an enzyme can be selected file 2: Figure S5). that confer herbicide resistance through a substitution in Herbicide resistance is an important goal in modern a single amino acid . Acetyl-coenzyme A carboxylase crop breeding as it will reduce the time cost for weeding. (ACC) is a key enzyme in lipid biosynthesis and it has In turn, this makes a significant contribution to increas- been shown that a T to C replacement (C2088R) in ing food productivity and reducing soil degradation. Lolium rigidum could endow plants with resistance to Herbicides often target specific enzymes in metabolic the herbicides across the aryloxyphenoxypropionate Li et al. Genome Biology (2018) 19:59 Page 5 of 9 Table 1 Mutation frequencies induced by PABE-7 in the T0 rice and wheat plants Species Target site sgRNA form Number of mutant Number of transgenic A Tto G C Genotype of mutations Heterozygous/ a b lines/plants rice lines or bombarded frequency (%) homozygous embryos of wheat Rice OsACC-T1 sgRNA 9 130 6.9 T >C (2); T T >C C (7) 9/0 4 4 4 7 4 7 esgRNA 33 160 20.6 T >C (10); T >C (2); T T >C C (21) 32/1 4 4 7 7 4 7 4 7 OsALS-T1 sgRNA 16 184 8.7 A >G (16) 16/0 5 5 esgRNA 42 196 21.4 A >G (1); A >G (41) 42/0 4 4 5 5 OsCDC48-T3 sgRNA 19 210 9.0 A >G (19) 19/0 5 5 esgRNA 60 180 33.3 A >G (60) 60/0 5 5 OsDEP1-T1 sgRNA 101 217 46.5 A >G (2); A >G (90); A A >G G (9) 88/13 4 4 6 6 4 6 4 6 esgRNA 83 211 39.3 A >G (4); A >G (73); A A >G G (6) 77/6 4 4 6 6 4 6 4 6 OsDEP1-T2 sgRNA 5 154 3.2 A >G (5) 5/0 6 6 esgRNA 34 215 15.8 A >G (1); A >G (32); 33/1 5 5 6 6 A A >G G (1) 3 6 3 6 OsNRT1.1B-T1 sgRNA 116 303 38.3 A >G (8); A >G (30); A A >G G (3); A A >G G (75) 111/5 6 6 8 8 4 8 4 8 6 8 6 8 esgRNA 149 252 59.1 A >G (6); A >G (46); A A >G G (2); A A >G G (95) 136/13 6 6 8 8 4 8 4 8 6 8 6 8 Wheat TaDEP1 esgRNA 5 460 1.1 A >G (4, AaBBDD; 5/0 8 8 1, AABbDD) TaGW2 esgRNA 2 480 0.4 A >G (2, AABbDD) 2/0 5 5 a b The number of mutant lines for rice and the number of mutant plants for wheat. Based on the number of T0 lines (rice) or plants (wheat) carrying the observed mutations over the total number of T0 transgenic rice lines analyzed or bombarded immature embryos of wheat Li et al. Genome Biology (2018) 19:59 Page 6 of 9 (APP), cyclohexanedione (CHD), and phenylpyrazoline Discussion (PPZ) chemical groups . The point mutation C2088R Despite the newly developed high efficiency of cytidine in Lolium rigidum corresponds to C2186R in rice (Oryza deaminase mediated C to T substitution exhibiting a sativa), which is our target site OsACC-T1. Examination great potential for disease therapeutic and agronomic of 160 pH-PABE-7-esgRNA-transformed lines revealed traits engineering , additional base editing tools are that 33 harbored at least one T to C substitution in the needed for expanding editing more DNA nucleotides. target region (mutation efficiency of 20.6%) (Table 1; Here, we adapted and optimized a plant ABE system Additional file 2: Table S1). One of the mutant lines con- (fusion of an evolved tRNA adenosine deaminase with tained a homozygous substitution (T T >C C ), whereas nuclease-inactivated CRISPR/Cas9) to efficiently and 4 7 4 7 the remaining 32 contained heterozygous substitutions: 20 specifically achieve targeted conversion of adenine to with double-base substitutions (T T >C C ), ten with guanine in crop plants. To our knowledge, this is the 4 7 4 7 T >C single-base substitutions, and two that first report of achieving wheat A to G base-edited plants 4 4 contained single-base substitutions providing the de- and herbicide-resistant rice plants with the plant ABE sired C2186R amino acid substitution at one of the system. High base-editing efficiency, low indels, and high alleles (T >C ;T0–7and T0–13) (Fig. 2e;Table 1; purity products make this plant ABE system outperform 7 7 Additional file 2: Figure S5b). We did not detect mutations HDR-mediated genome editing. in the potential off-target regions among all OsACC-T1 Based on the ABE7.10 architectures for human cells, mutant lines (Additional file 2: Tables S2 and S3). We then we optimized the system for crop plants from two per- assessed the herbicide resistance of the T0–13 mutant car- spectives. One was by optimizing the position of the rying the heterozygous C2186R substitution. After one tRNA adenosine deaminase relative to the nCas9, and week of growth on the regeneration medium supplemented the number and locations of NLSs. Our observation with 0.086 ppm haloxyfop-R-methyl, the mutant plant had shows that placing the ecTadA-ecTadA* adenosine normal phenotypes with no symptoms of damage whereas deaminase at the N-terminus of nCas9 and the presence wild-type (WT) plants displayed severe stunting and with- of three NLSs at the C-terminus (PABE-7) maximizes ered leaves (Fig. 2e). To the best of our knowledge, this is editing efficiency, probably because this configuration the first report of producing C2186R substitution of resist- maximizes fusion protein folding and nuclear importing. ant rice plants using genome editing tools. The other improvement to the plant ABE system was We also used the plant ABE system to generate based on comparing three forms of sgRNA (native base-edited plants in wheat by targeting TaDEP1 and sgRNA, esgRNA, and tRNA-sgRNA). We found that the TaGW2 genes. PABE-7 and pTaU6-esgRNA constructs esgRNA showed a higher editing efficiency than the na- (Additional file 2: Figure S1e and Table S1) were delivered tive sgRNA and the tRNA-sgRNA in both protoplasts into immature wheat embryos by particle bombardment, and regenerated plants, indicating that the esgRNA has and plants regenerated without herbicide selection, as a higher expression level and better binding activity with previously described . Through T7E1 and Sanger Cas9 . With our most effective combination, PABE-7 sequencing, we obtained five A to G heterozygous plus esgRNA, we obtained base-edited rice and wheat 8 8 TaDEP1 mutant plants regenerated from 460 bombarded plants in the T0 generation. The herbicide-resistant rice immature embryos (Table 1;Additional file 2:FigureS6a), plants harboring the C2186R substitution in OsACC was with four mutants heterozygous for TaDEP1-A (tade- also obtained, indicating this plant ABE system is a reli- p1-AaBBDD) and one mutant heterozygous for TaDEP1-B able tool for achieving targeted base editing in crop (tadep1-AABbDD)(Table 1;Additional file 2:Figure S6a). plants. For the TaGW2 target site, two heterozygous mutants There are still opportunities for extending and optimiz- were identified. Both harbored an A to G substitution at ing the plant ABE system. One could use engineered Cas9 position 5 for TaGW2-B (tagw2-AABbDD)(Table 1; variants with different protospacer-adjacent motif (PAM) Additional file 2: Figure S6b). Again, no indels were specificities (xCas9, SpCas9-VQR, SpCas9-VRER, SaCas9, observed in the target region of all mutant plants. Further- and SaCas9-KKH), or Cpf1 [9, 18, 19], to expand the more, PCR screening with six primer sets, specific for number of sites that can be targeted. The plant ABE sys- PABE-7 and pTaU6-esgRNA (Additional file 2:FigureS7a tem combined with the plant C to T base editing system and Table S3), confirmed that three of five TaDEP1 by ligating sgRNA with different aptamers (MS2, PP7, mutants and two TaGW2 mutants did not carry the COM, and boxB) [20, 21] could achieve simultaneous A transgene vectors (Additional file 2: Figure S7b). Taken to- to G and C to T changes, and could be used to correct gether, these results support that the plant ABE system is point mutations related to important agronomic traits. It effective in inducing specific point mutations in rice and could also provide a novel forward genetics tool to screen wheat in a highly specific and precise manner without gain-of-function and partial loss-of-function genetic vari- causing other genomic modifications. ants at the resolution of single bases. Furthermore, plant Li et al. Genome Biology (2018) 19:59 Page 7 of 9 ABE ribonucleoproteins (RNPs) could be delivered to deep amplicon sequencing and T7E1 and PCR restric- create transgene-free mutant plants, which could tion enzyme digestion assays (PCR-RE assays; see avoid inserting recombinant DNA into host genomes, below). and would have a good chance of being commercial- ized [17, 22]. DNA extraction Genomic DNA was extracted with a DNA quick Plant Conclusions System (TIANGEN BIOTECH, Beijing, China). The tar- We describe here an efficient plant base-editing system geted site was amplified with specific primers, and the that induces precise A� TtoG� C substitutions across a amplicons were purified with an EasyPure PCR Purifica- broad range of endogenous genomic loci. The effective tion Kit (TransGen Biotech, Beijing, China), and quanti- deamination window of this plant ABE system extends fied with a NanoDrop™ 2000 Spectrophotometer (Thermo from positions 4 to 8 of the protospacer and produces Fisher Scientific, Waltham, MA, USA). high-fidelity substitutions at the targeted loci with low indels. These findings, together with previously de- Next-generation sequencing scribed plant substitution systems [23–26], extend the Genomic DNA extracted from the desired protoplast application of base editing to the majority of codons and samples at 60 h post-transfection was used as template. now provides feasible opportunities for significant in In the first round PCR, the target region was amplified vivo mutagenesis studies and trait improvement in using site-specific primers (Additional file 2: Table S3). plants. In the second round PCR, both forward and reverse bar- codes were added to the ends of the PCR products for li- Methods brary construction (Additional file 2: Table S3). Equal Plasmid construction amounts of the PCR products were pooled and samples To construct vectors PABE-1 to PABE-7, the tRNA editing were sequenced commercially (Mega Genomics, Beijing, deaminase ecTadA, ecTadA*, 32aa linker, and nCas9 (D10A) China) using the Illumina NextSeq 500 platform. The sequences were codon-optimized for cereal plants, and syn- sgRNA target sites in the sequenced reads were exam- thesized commercially (GENEWIZ, Suzhou, China). The ined for A to G substitutions and indels. The amplicon various combinations of the ecTadA-ecTadA* and nCas9 fu- sequencing was repeated three times for each target site, sion protein sequences were cloned into the vector pJIT163 using genomic DNA extracted from three independent backbone. The native constructs pOsU3-sgRNA and protoplast samples. pTaU6-sgRNA, tRNA-sgRNA of pOsU3-tRNA-sgRNA and pTaU6-tRNA-sgRNA were made as previously described Agrobacterium-mediated transformation of rice callus cells [14, 27, 28]; the esgRNA of pOsU3-esgRNA and Agrobacterium tumefaciens strain AGL1 was transformed pTaU6-esgRNA were synthesized commercially (GENEWIZ, with the pH-PABE-7-esgRNA or pH-PABE-7-sgRNA Suzhou, China). To construct the pH-PABE-7-esgRNA and binary vectors by electroporation. Agrobacterium-me- pH-PABE-7-sgRNA binary vector, PABE-7 and esgRNA or diated transformation of callus cells of Zhonghua11 was sgRNA expression cassettes were cloned into the pHUE411 conducted as reported . Hygromycin (50 μg/ml) was backbone . Point mutations were introduced into the used to select transgenic plants. coding sequence of GFP with the Fast Mutagenesis System (TransGen Biotech, Beijing, China), yielding expression cas- Biolistic delivery of DNA constructs into wheat immature settesproducing mGFP.All theprimersetsusedinthiswork embryo cells are listed in Additional file 2: Table S3 and were synthesized The plasmid DNAs of PABE-7 and pTaU6-esgRNA were by Beijing Genomics Institute (BGI). simultaneously delivered into the immature embryos of Kenong199 via particle bombardment as previously de- Protoplast transfection scribed . After the bombardment, the embryos were We used the winter wheat variety Kenong199 and the cultured for plantlet regeneration on the media without Japonica rice variety Zhonghua11 to prepare the proto- a selective agent . plasts used in this study. Protoplast isolation and trans- formation were performed as previously described [27, Mutant identification by T7E1 and PCR-RE assays and 28]. Plasmid DNA (10 μg per construct) was introduced Sanger sequencing into the desired protoplasts by PEG-mediated transfec- T7E1 and PCR-RE assays and Sanger sequencing were tion, the mean transformation efficiency being 45–60% performed to identify rice and wheat mutants with A to by flow cytometry (FCM). The transfected protoplasts G conversions in target regions, as described previously were incubated at 23 °C. At 60 h post-transfection, the [27, 28]. For rice, the T0 transgenic plants were exam- protoplasts were collected to extract genomic DNA for ined individually. For wheat, plantlets (usually 3–4) Li et al. Genome Biology (2018) 19:59 Page 8 of 9 derived from each bombarded immature embryo were Received: 3 April 2018 Accepted: 3 May 2018 pooled for the assays, and the positive pools were exam- ined further to identify individual mutant plantlets . References A to G mutation frequencies were calculated from band 1. Yin K, Gao C, Qiu JL. Progress and prospects in plant genome editing. Nat intensities measured with UVP VisionWorks LS Image Plants. 2017;3:17107. Acquisition Analysis Software 7.0, as described . 2. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–21. 3. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Detection of off-target mutations Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, et al. 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Published: May 29, 2018