Generation of Targeted Point Mutations in Rice by a Modified CRISPR/Cas9 System (2016)

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated Cas9 endonuclease)-mediated genome editing has revolutionized biological research and crop improvement because of its specificity, simplicity, and versatility (reviewed in Komor et al., 2016aKomor A.C. Badran A.H. Liu D.R. CRISPR-based technologies for the manipulation of eukaryotic genomes.Cell. 2016; https://doi.org/10.1016/j.cell.2016.10.044Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar). Editing a gene by CRISPR/Cas9 only has three requirements: (1) expression of the nuclear localized Cas9 protein; (2) production of a guide RNA (gRNA) molecule, whose first 20 nucleotides are complementary to the target gene; (3) the NGG PAM site that is located immediately adjacent to the 3′ end of the target sequence (Figure 1A). In the presence of a gRNA, Cas9 generates a double-strand break (DSB) at the target sequence. DSB repair by the error-prone, non-homologous end-joining (NHEJ) mechanism often leads to small deletions or insertions at the target site, providing a valuable venue to generate knockout and loss-of-function mutants. The majority of the reported CRISPR/Cas9-mediated gene editing in plants belongs to this category (Miao et al., 2013Miao J. Guo D. Zhang J. Huang Q. Qin G. Zhang X. Wan J. Gu H. Qu L.J. Targeted mutagenesis in rice using CRISPR-Cas system.Cell Res. 2013; 23: 1233-1236Crossref PubMed Scopus (585) Google Scholar, Xie and Yang, 2013Xie K. Yang Y. RNA-guided genome editing in plants using a CRISPR-Cas system.Mol. Plant. 2013; 6: 1975-1983Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, Ma et al., 2015Ma X. Zhang Q. Zhu Q. Liu W. Chen Y. Qiu R. Wang B. Yang Z. Li H. Lin Y. et al.A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants.Mol. Plant. 2015; 8: 1274-1284Abstract Full Text Full Text PDF PubMed Scopus (1160) Google Scholar, Wang et al., 2015Wang Z.P. Xing H.L. Dong L. Zhang H.Y. Han C.Y. Wang X.C. Chen Q.J. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation.Genome Biol. 2015; 16: 144Crossref PubMed Scopus (527) Google Scholar, Char et al., 2016Char S.N. Neelakandan A.K. Nahampun H. Frame B. Main M. Spalding M.H. Becraft P.W. Meyers B.C. Walbot V. Wang K. et al.An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.Plant Biotechnol. J. 2016; https://doi.org/10.1111/pbi.12611Crossref PubMed Scopus (185) Google Scholar, Gao et al., 2016Gao X. Chen J. Dai X. Zhang D. Zhao Y. An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing.Plant Physiol. 2016; 171: 1794-1800Crossref PubMed Scopus (153) Google Scholar). While such mutants are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by single-nucleotide polymorphisms or by dominant gain-of-function point mutations. Homology-directed repair (HDR) of the DSB at specific locations can provide a feasible approach to achieve gene replacement. However, it has been very challenging to make use of HDR in plants to introduce point mutations because of the very low frequency of targeted integration. So far only a few special cases of CRISPR/Cas9-mediated HDR have been reported in plants (Sun et al., 2016Sun Y.W. Zhang X. Wu C.Y. He Y. Ma Y.Z. Hou H. Guo X.P. Du W.M. Zhao Y.D. Xia L.Q. Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase.Mol. Plant. 2016; 9: 628-631Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). NHEJ-mediated repair may also be used to introduce point mutations through gene replacement if the target sequences of CRISPR/Cas9 are located in introns (Li et al., 2016Li J. Meng X. Zong Y. Chen K. Zhang H. Liu J. Li J. Gao C. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.Nat. Plants. 2016; 2: 16139Crossref PubMed Scopus (228) Google Scholar). Nevertheless, it is still a major challenge to generate targeted point mutations in plants. Cas9 has been engineered as a DNA-binding protein without the nuclease activities (dCas9), which has D10A and H840A mutations. A gRNA molecule brings dCas9 protein to a specific DNA target, thus offering unprecedented opportunities to modify structures or properties of DNA/chromatin near the target site (Komor et al., 2016aKomor A.C. Badran A.H. Liu D.R. CRISPR-based technologies for the manipulation of eukaryotic genomes.Cell. 2016; https://doi.org/10.1016/j.cell.2016.10.044Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar). Fusions of dCas9 with transcription activator or repressors have led to activation or repression of specific gene expression. A methyltransferase fusion with dCas9 caused modifications of histones in a gRNA-specific manner (Komor et al., 2016aKomor A.C. Badran A.H. Liu D.R. CRISPR-based technologies for the manipulation of eukaryotic genomes.Cell. 2016; https://doi.org/10.1016/j.cell.2016.10.044Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar). Recently, dCas9 fused with a cytidine deaminase enzyme was shown to have the ability to convert C to T (or G to A) within a window of approximately five nucleotides, providing a very useful tool to introduce point mutations at specific locus defined by a gRNA molecule (Komor et al., 2016bKomor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (2385) Google Scholar). Moreover, the system was further improved by combining a nicked Cas9 with only the D10A mutation (nCas9), a cytidine deaminase enzyme, and the uracil glycosylase inhibitor (UGI) that inhibits base-excision repair (Komor et al., 2016bKomor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (2385) Google Scholar). The improved version was named as BE3 (APOBEC1-XTEN-nCas9-UGI, the third generation of base editor, BE3) (Supplemental Figure 1). The complete sequence of BE3 unit, which is also available at https://www.addgene.org/73021/sequences/#depositor-full, targets the non-complementary strand for cleavage and confers desired base-editing outcomes through manipulating the cellular DNA repair response (Figure 1A). BE3 had 15%–75% base-editing efficiency in animal cells with very minimal indel formation (Komor et al., 2016bKomor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (2385) Google Scholar). Therefore, base editing expands the scope, ability, and efficiency of CRISPR/Cas9-mediated genome editing for introducing point mutations. However, it is not clear whether similar strategies will work in plant species, especially in crops. In this study, we report the use of nCas9 fused with a cytidine deaminase enzyme and the UGI to generate targeted point mutations in rice. We cloned the BE3 cassette to our pCXUN vector to generate pCXUN-BE3, which has the ability to target a specific locus in genome when a gRNA molecule is simultaneously expressed (Supplemental Figure 2). We used the maize (Zea mays) ubiquitin promoter to express BE3. We then inserted the expression cassette of a gRNA, which was under the control of the rice U3 promoter, into the PmeI site of pCXUN-BE3 (Supplemental Figure 2). We tested our base-editing vectors in rice for their feasibility and efficacy. Three targets were chosen: one target (P2) in the OsPDS, which encodes a phytoene desaturase, and two targets (S3 and S5) in the OsSBEIIb, which encodes a starch branching enzyme IIb in rice (Supplemental Table 1). We delivered the vectors into rice calli through Agrobacterium-mediated transformation. For target S5 (Figure 1B), we transformed 93 calli and obtained 52 independent lines after two rounds of hygromycin selection and regeneration. We performed PCR amplification using the primer sets designed to specifically amplify nCas9, gRNAs, and hptII sequences, respectively (Supplemental Table 2). Results showed that all of the candidate plants tested contained nCas9, gRNAs, and hptII (Supplemental Figure 3). PCR/restriction enzyme (RE) analyses (see primers in Supplemental Table 2) were then used to screen for plants with targeted mutations (Figure 1B). Once the expected substitution of G to A in the target region (marked red in Figure 1B) takes place in S5 in OsSBEIIb gene, the BstNI restriction site will be disrupted. From 52 independent plants, we identified 23 lines that had BstNI-resistant bands (Figure 1B), suggesting that the mutagenesis efficiency is more than 40%. When the PCR products of target S5 were digested by BstNI, wild-type would produce two fragments with sizes of 233 bp and 83 bp, respectively (Figure 1B). Putative mutants had either undigested (homozygous or bi-allelic) or three fragments of 316 bp, 233 bp, and 83 bp (heterozygous) (Figure 1B). We further sequenced all of the samples with BstNI-resistant bands and the results indicated that there were three groups of mutations (Figure 1C and 1D). The first group had 10 lines with the expected G to A changes at positions 5 and 6 (G5 to A5 and G6 to A6) (Figure 1C). Among these, three were homozygous (S5-17, S5-36, and S5-46), and seven were either heterozygous (S5-1, S5-8, S5-21, S5-33, S5-42, and S5-43) or bi-allelic line (S5-34). The first group constitutes 43% (10/23) of the mutants we had obtained for the S5 target, and this group of mutants had the exact mutations that we expected. For this particular target, the overall efficiency of obtaining the desired mutations is about 20%, which is much higher than HDR-mediated or intron-targeted NHEJ-mediated gene replacement (Li et al., 2016Li J. Meng X. Zong Y. Chen K. Zhang H. Liu J. Li J. Gao C. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.Nat. Plants. 2016; 2: 16139Crossref PubMed Scopus (228) Google Scholar, Sun et al., 2016Sun Y.W. Zhang X. Wu C.Y. He Y. Ma Y.Z. Hou H. Guo X.P. Du W.M. Zhao Y.D. Xia L.Q. Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase.Mol. Plant. 2016; 9: 628-631Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). The second group contained the expected G to A changes, but also had either G to C or G to T conversions at positions 5 and 6 (Figure 1C). It is not clear how the unintended base conversions took place. This group had eight lines with one homozygous (S5-26) and seven heterozygous (S5-10, S5-25, S5-44, S5-45, S5-48, S5-50, and S5-52) (Figure 1C). The third group had five lines that contained either bi-allelic (S5-18, S5-31, and S4-47) or heterozygous (S5-16 and S5-23) mutations, which were unexpected random deletions or insertions at the target sites. These indels were probably caused by NHEJ-mediated DNA repair. We noticed that we only observed the targeted mutations in the intended two Gs (Figure 1B and 1C). There are five other Gs in the target region, but none of them were mutated in our identified mutants (Figure 1C). One reason for this could be that we only sequenced the BstNI-resistant plants, suggesting that the observed base-editing efficiency is underestimated. Another reason is that some of the Gs might be outside the editing window because the efficient deamination window is approximately 5 nt from positions 4 to 8 (Komor et al., 2016bKomor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (2385) Google Scholar). To further test the efficacy of our vectors in base editing in rice, we targeted another site (S3) in the OsSBEIIb (Supplemental Figure 4), to enable comparison of the effects of target sequences. For S3 target, we transformed 64 calli and obtained 38 independent transgenic lines. Similar to the case of S5 (Figure 1), we also obtained three groups of mutations (Supplemental Figure 4). The first group included four lines with the expected C to T conversion, of which three were homozygous at position 7 and one was heterozygous with three mutations of C to T at positions 1, 2, and 7 in one DNA strand (Supplemental Figure 4). The second group only had one bi-allelic mutation with C to T at position 7 in one DNA strand, and C to G in another strand at position 7. The third group had six lines with unexpected mutations, four of which were homozygous with C to G conversion at position 7, and two of which were bi-allelic with one strand that had C to G conversion at position 7 and another with C to G conversions at both positions 1 and 7 (Supplemental Figure 4). We also targeted the OsPDS gene (Supplemental Figure 5). We obtained 88 transgenic plants out of the 99 transformed calli. Interestingly, only two plants had EcoRI-resistant bands (Supplemental Figure 5). For line P2-21, both the G8 and G10 were converted into A and the line was heterologous. Another mutant line, P2-79, had a G to C change at position 8 (Supplemental Figure 5). Taking the results of S5 and S3 targets together, we propose that base-editing efficiency is dependent on the target sequence. It will be interesting to further investigate whether the target selection criteria for conventional CRISPR/Cas9-mediated gene editing is also suitable for screening targets for base editing. In summary, we have successfully introduced precise point mutations to three target sites in rice, thus providing a feasible and effective tool for targeted base editing in a crop plant. Base editing offers higher efficiency in targeted base correction than HDR-mediated gene replacement. Moreover, it is much easier to construct a base-editing vector than generate an HDR plasmid. Therefore, the vectors and methods described here offer great potential for crop genetic improvement and breeding. This work is partly funded by the Ministry of Science and Technology of China (grant nos. 2016YFD0102003 and 2016YFD0100500) and the Ministry of Agriculture of China (grant no. 2016ZX08010-003). Y.Z. is supported by a start-up fund from the National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University. Y.S. is supported by a GSCAAS-ULg Joint PhD Program (grant no. MOE11BE1A20131371N).