Expanding base editing scope to near-PAMless with engineered CRISPR/Cas9 variants in plants

Abstract

Base editors (BEs), including cytosine base editor (CBE) and adenine base editor (ABE), have been widely used to generate irreversible nucleotide substitution in plants and animals. However, their wide applications are largely hindered by the strict NG protospacer adjacent motif (PAM) sequences recognized by Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants, such as SpCas9-NG and xCas9 (Hua et al., 2019Hua K. Tao X. Han P. Wang R. Zhu J.K. Genome engineering in rice using Cas9 variants that recognize NG PAM sequences.Mol. Plant. 2019; 12: 1003-1014Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar; Ren et al., 2019Ren B. Liu L. Li S. Kuang Y. Wang J. Zhang D. Zhou X. Lin H. Zhou H. Cas9-NG greatly expands the targeting scope of the genome-editing toolkit by recognizing NG and other atypical PAMs in rice.Mol. Plant. 2019; 12: 1015-1026Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar; Wu et al., 2019Wu Y. Xu W. Wang F. Zhao S. Feng F. Song J. Zhang C. Yang J. Increasing cytosine base editing scope and efficiency with engineered cas9-PmCDA1 fusions and the modified sgRNA in rice.Front. Genet. 2019; 10: 379Crossref PubMed Scopus (22) Google Scholar; Zhong et al., 2019Zhong Z. Sretenovic S. Ren Q. Yang L. Bao Y. Qi C. Yuan M. He Y. Liu S. Liu X. et al.Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-targeting Cas9-NG.Mol. Plant. 2019; 12: 1027-1036Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar; Zhang et al., 2020Zhang C. Xu W. Wang F. Kang G. Yuan S. Lv X. Li L. Liu Y. Yang J. Expanding the base editing scope to GA and relaxed NG PAM sites by improved xCas9 system.Plant Biotechnol. J. 2020; 18: 884-886Crossref PubMed Scopus (9) Google Scholar). Most recently, it was reported that three new SpCas9 variants, SpCas9-NRRH, SpCas9-NRTH, and SpCas9-NRCH, could recognize non-G PAMs (NRNH, where R is A or G and H is A, C, or T) in human cells (Miller et al., 2020Miller S.M. Wang T. Randolph P.B. Arbab M. Shen M.W. Huang T.P. Matuszek Z. Newby G.A. Rees H.A. Liu D.R. Continuous evolution of SpCas9 variants compatible with non-G PAMs.Nat. Biotechnol. 2020; 38: 471-481Crossref PubMed Scopus (93) Google Scholar). Meanwhile, SpRY, another new SpCas9 variant, was developed to greatly expand the editing scope of BEs to nearly PAMless (Walton et al., 2020Walton R.T. Christie K.A. Whittaker M.N. Kleinstiver B.P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.Science. 2020; 368: 290-296Crossref PubMed Scopus (223) Google Scholar). In this study, we generated a series of efficient BE toolkits and almost achieved C-to-T mutation without PAM restriction except for NTG PAM, and largely expanded A-to-G mutation scope in stable transformed rice, providing a reference for application in other plants. Given the broad PAM compatibility of near-PAMless SpRY and its good performance in human cells, we speculated that it could be also utilized to expand BEs' editing scope in rice. We first applied it in a CBE system. The D10A nickase of the plant-favored codon-optimized SpRY (SpRYn) (Supplemental Sequence 1) was fused to the N terminus of Petromyzon marinus cytidine deaminase1 (PmCDA1) with two copies of uracil DNA glycosylase inhibitor (UGI), which connected to hygromycin phosphotransferase (HPT) by a P2A, a self-cleaving 2A peptide. This fusion protein was driven by the Ubiquitin promoter of rice (OsUbi). tRNA together with enhanced single guide RNA (esgRNA) was placed under control of the rice U6 promoter. Targets were finally added using the method described by 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/Cas9n 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 (870) Google Scholar. This editor was designated as SpRYn-CBE (Figure 1A). Considering the robust activity of SpRY on NRN (where R is A or G) PAM sites in human cells, we first chose 12 NAN PAM sites from six different rice genes for assessment (Figure 1B and Supplemental Table 1). In T0 plants, SpRYn-CBE displayed robust base editing activity on two NAC and two NAG PAM sites with editing efficiencies of 62.2%, 13.2%, 38.1%, and 45.2%, respectively (Figure 1B and Supplemental Figure 1). However, no lines were edited at four NAA PAM sites (NAA-C1 to NAA-C4) and four NAT PAM sites (NAT-C1 to NAT-C4), unlike its good performance in human cells (Walton et al., 2020Walton R.T. Christie K.A. Whittaker M.N. Kleinstiver B.P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.Science. 2020; 368: 290-296Crossref PubMed Scopus (223) Google Scholar). Another four NAA (NAA-C5 to NAA-C8) and four NAT (NAT-C5 to NAT-C8) PAM sites were tested for further identification, but none of them were mutated either (Supplemental Tables 1 and 2). Since the NAA and NAT PAMs could be well recognized by SpNRRH and SpNRTH, respectively in human cells (Miller et al., 2020Miller S.M. Wang T. Randolph P.B. Arbab M. Shen M.W. Huang T.P. Matuszek Z. Newby G.A. Rees H.A. Liu D.R. Continuous evolution of SpCas9 variants compatible with non-G PAMs.Nat. Biotechnol. 2020; 38: 471-481Crossref PubMed Scopus (93) Google Scholar), we used SpNRRHn-CBE and SpNRTHn-CBE to further test NAA and NAT PAM sites in rice (Figure 1A). SpNRRHn-CBE showed superior editing efficiencies of more than 90% on three NAA PAM sites (NAA-C2 to NAA-C4) and a moderate efficiency of 39.1% on target NAA-C1 in T0 plants (Figure 1B and Supplemental Figure 2). SpNRTHn-CBE also enabled effective editing activity on two NAT PAM sites with frequencies of 18.8% and 87.5%, respectively (Figure 1B and Supplemental Figure 2). Collectively, these results demonstrate that SpRYn-CBE (NAC, NAG) together with SpNRRHn-CBE (NAA) and SpNRTHn-CBE (NAT) could expand the targeting scope of CBE to NAN PAM sites in rice. Next, we evaluated the activity of SpRYn-CBE at eight target sites harboring NGN PAMs from six different rice genes (Figure 1C and Supplemental Table 1). SpRYn-CBE showed detectable base mutations at all sites, with frequencies ranging from 4.2% to 37.1% (Figure 1C and Supplemental Figure 1), which meant that SpRYn-CBE was also an effective cytosine base editor for genomic sites with NG PAM in rice, just like SpCas9-NG involved CBE. As SpCas9-NG possessed lower activity on NGC PAM sites in human cells and rice (Nishimasu et al., 2018Nishimasu H. Shi X. Ishiguro S. Gao L. Hirano S. Okazaki S. Noda T. Abudayyeh O.O. Gootenberg J.S. Mori H. et al.Engineered CRISPR-Cas9 nuclease with expanded targeting space.Science. 2018; 361: 1259-1262Crossref PubMed Scopus (381) Google Scholar; Ren et al., 2019Ren B. Liu L. Li S. Kuang Y. Wang J. Zhang D. Zhou X. Lin H. Zhou H. Cas9-NG greatly expands the targeting scope of the genome-editing toolkit by recognizing NG and other atypical PAMs in rice.Mol. Plant. 2019; 12: 1015-1026Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), we generated SpCas9n-NG-CBE (Supplemental Figure 3) and compared its activity on NGC sites with that of SpRYn-CBE. Both NGC-C1 and NGC-C2, which could be mutated by SpRYn-CBE, were not edited with SpCas9n-NG-CBE (Supplemental Table 2). Another three NGC sites (NGC-C3 to NGC-C5) were then selected for further comparison (Supplemental Table 1). SpRYn-CBE could induce base mutations at all three sites, whereas SpCas9n-NG-CBE did not induce base conversion except at the NGC-C5 site (Supplemental Table 2). These results suggest that for NGC PAM sites, SpRYn-CBE outperforms SpCas9n-NG-CBE and might act as a good alternative to SpCas9n-NG-CBE in rice. Additionally, we also assessed the activities of SpRYn-CBE and SpCas9n-CBE (Supplemental Figure 3) on NGG PAM sites. We found that SpRYn-CBE showed editing activity comparable with that of SpCas9n-CBE (Figure 1C and Supplemental Table 2). For NYN (Y = C or T) PAMs, we tested the editing activity of SpRYn-CBE on five NCN PAM sites and six NTN PAM sites (Supplemental Table 1). SpRYn-CBE enabled efficient editing across all NCN and NTH (H = A, C, or T) PAM sites, showing editing efficiencies of 55.2% (NCA-C1), 7.5% (NCT-C1), 9.3% (NCC-C1), 20.7% (NCG-C1), 36.6% (NCG-C2), 13.5% (NTA-C1), 10.5% (NTT-C1), and 15.4% (NTC-C1) (Figure 1C and Supplemental Figure 1). However, no base mutations were detected at three NTG PAM sites (Supplemental Table 2). These results indicate that SpRYn-CBE could expand the targeting scope to those sites with NCN and NTH PAMs. Taken together, the above results demonstrate that SpRYn-CBE is capable of editing target sites with nearly all PAMs in rice. Based on such a good performance of SpRY in CBE, we further evaluated the function of SpRY in expanding targeting scope of ABE. The SpRYn together with HPT was fused to the C terminus of Escherichia coli tRNA adenosine deaminase variant (ecTadA∗) to generate SpRYn-ABE (Figure 1D). Sixteen NAN, 12 NGN, 10 NCN, and 9 NTN PAM sites were chosen for evaluation (Supplemental Table 3). In T0 plants, SpRYn-ABE could efficiently edit all selected sites with NAT, NAC, or NAG PAMs, with frequencies from 4.2% to 85% (Figure 1E and Supplemental Figure 4). Unexpectedly, sites with NAA PAMs could still not be targeted, which was quite different from the results in human cells (Supplemental Table 4). We also could not detect any mutations at another four NAA PAM sites (NAA-A5 to NAA-A8) (Supplemental Tables 3 and 4). For NGN PAMs, SpRYn-ABE was capable of efficiently targeting sites with NGV (V = A, C, or G) PAMs, achieving editing efficiencies of 16.7% (NGA-A1), 11.1%–46.7% (NGC-A1 to NGC-A3), and 4.8%–19.1% (NGG-A1 to NGG-A4) (Figure 1E and Supplemental Figure 4), but for four NGT PAM sites no mutation was detected (Supplemental Table 4). Across the sites with NYN PAMs, the sites bearing NCA, NCG, NTT, and NTG PAMs could be edited with frequencies of 10.5%, 9.5%, 29.2%, and 26.7%, respectively in T0 plants using SpRYn-ABE (Figure 1E and Supplemental Figure 4). However, all tested sites with other NYN PAMs, including NCT, NCC, NTA, and NTC, failed to be edited (Supplemental Table 4). Taken together, these results suggest that SpRYn-ABE could expand the editing scope to NAB (B = C, T, or G), NCR (R = A or G), and NTK (K = T or G) PAM sites in rice. To address the limitation of NAA and NGT PAMs in the SpRYn-ABE system, we generated SpNRRHn-ABE (Figure 1D) and SpGn-ABE (Supplemental Figure 3). Unexpectedly, they still failed to edit the target sites (Supplemental Table 4), although SpNRRHn-CBE worked well. This might be caused by the difference between ABE and CBE systems. In addition, we compared the editing activity between SpRYn-ABE and SpCas9n-NG-ABE (Supplemental Figure 3) on those sites with NGC PAMs and found that SpCas9n-NG-ABE could not edit NGC-A2 and NGC-A3 at all, and the editing efficiency of 9.1% on NGC-A1 was greatly lower than that by SpRYn-ABE (46.7%) (Figure 1E and Supplemental Table 4). These results suggest that SpRYn-ABE could be a good alternative to SpCas9n-NG-ABE in rice, especially for NGC PAM sites. Considering the low efficiencies on NGG PAM sites using SpRYn-ABE, we tested SpCas9n-ABE (Supplemental Figure 3) and found it outperformed SpRYn-ABE, which meant SpCas9n-ABE was preferred for sites harboring NGG PAMs (Figure 1E and Supplemental Table 4). All 190 and 101 edited T0 plants were taken together to investigate the technical parameters of SpRYn-CBE and SpRYn-ABE, respectively. The editing window of SpRYn-CBE spanned positions 1 to 13 within the protospacer, with the editing preference of C3 > C2 > C4 > C5 ≫ Cs from other positions (Figure 1F). The editing window of SpRYn-ABE spanned positions 2 to 14 (Figure 1G). A4, A5, A6, A7, A8, and A14 were edited more frequently (A7 > A5 ≈ A6 > A8 > A4 > A14), whereas A2, A3, A9, and A11 were mutated relatively infrequently (less than 10%) (Figure 1G), which was different from that in SpRYn-CBE. Their editing windows were wider than those in the reported SpRY CBEmax (3–16) and SpRY ABEmax (3–10) in human cells (Walton et al., 2020Walton R.T. Christie K.A. Whittaker M.N. Kleinstiver B.P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.Science. 2020; 368: 290-296Crossref PubMed Scopus (223) Google Scholar). For mutation type, single C-to-T or A-to-G mutants were predominant (Supplemental Table 5). Single C or A mutation could be produced at the position with the highest editing efficiency at each target in T0 plants (Supplemental Table 5). Homozygous mutations and Indels were seldom generated (Supplemental Table 5). Although limited edited target sites were analyzed for SpNRRHn-CBE and SpNRTHn-CBE, they have technical parameters similar to those of SpRYn-CBE, except for more double C-to-T mutants and homozygous mutations (Supplemental Table 5). In the T-DNA region, a GTT motif was located immediately at the 3′ end of the target sites, which might be edited by both SpRYn-CBE and SpRYn-ABE. Most target sites were tested for their self-editing capability in T-DNA. Among those ineffective target sites, one out of nine and 3 out of 18 targets had T-DNA mutations produced by SpRYn-CBE and SpRYn-ABE, respectively (Supplemental Table 6). SpNRRHn-ABE and SpGn-ABE showed similar results (Supplemental Table 6). Among 29 edited target sites, only 15 sites showed self-editing by SpRYn-CBE (6/12) and SpRYn-ABE (9/17), and there was no direct relationship between genome editing efficiency and the occurrence of self-editing (Supplemental Table 6). However, it seemed that more efficient targets were more likely to be self-edited (Supplemental Table 6). SpNRRHn-CBE and SpNRTHn-CBE showed similar results (Supplemental Table 6). In addition, among the aforementioned 15 self-edited target sites, we found that all plants with genomic base mutations in 12 target sites were mutated in the T-DNA region, which was consistent with the results of Qin et al., 2020Qin R. Li J. Liu X. Xu R. Yang J. Wei P. SpCas9-NG self-targets the sgRNA sequence in plant genome editing.Nat. Plants. 2020; 6: 197-201Crossref PubMed Scopus (17) Google Scholar, and the average self-editing efficiency was higher than that in wild-type plants (Supplemental Table 6). All these data indicate that self-editing is more likely to occur at target sites with higher genome editing efficiency and at T0 plants with genomic base mutations, but there is no evidence that it could influence the genome editing efficiency of SpRYn-CBE and SpRYn-ABE. Considering the ability of SpRYn-CBE and SpRYn-ABE to edit sites with more PAMs, we speculated that the off-target activity might increase. Four target sites with relatively high editing efficiency were chosen from each NN PAM site for testing the off-target activity of SpRYn-CBE and SpRYn-ABE, respectively. Unexpectedly, for SpRYn-CBE, no mutations were identified at all tested potential off-target sites of NAC-C1, NTC-C1, and NGG-C3 in T0 plants with on-target base mutations (Supplemental Table 7). Only one C-to-T substitution and one deletion plant were identified at 17 potential off-target sites of NCA-C1 (Supplemental Table 7). Compared with SpRYn-CBE, SpRYn-ABE produced more off-target mutations and had higher mutation frequency (Supplemental Table 7). In summary, SpRYn-CBE could expand the targeting scope to those sites with NCN, NTH, NAG, and NAC PAMs. Together with SpNRRHn-CBE (NAA) and SpNRTHn-CBE (NAT), the targeting scope of CBE could be expanded to near-PAMless except for the only NTG PAM in rice. It is worth mentioning that SpRYn-CBE showed higher or comparable editing activity on NGC PAM and NGG PAM when compared with SpCas9n-NG-CBE and SpCas9n-CBE. As for ABE, SpRY also greatly expanded PAMs to NAB (B = C, T, or G), NCR (R = A or G), NTK (K = T or G), and NGV (V = A, C, or G) PAM in rice. Considering the outperformance of SpRYn-ABE, it could be a good alternative to SpCas9n-NG-ABE, although not for SpCas9n-ABE in rice. Therefore, SpRYn-CBE and SpRYn-ABE together with their clear technical parameters will sharply simplify CBE and ABE toolkits for further application in plants. These simplified toolkits should also be applied in maize, wheat, or other crops in the future. Further work can be carried out to employ more SpCas9 variants or Cas9 orthologs to cover the remaining PAMs. In addition, combination of the DisSUGs technology with the base editor toolkits listed herein may further improve the efficiency of these editors by enhancing the screening of plant base-edited cells (Xu et al., 2020Xu W. Yang Y. Liu Y. Kang G. Wang F. Li L. Lv X. Zhao S. Yuan S. Song J. et al.Discriminated sgRNAs-based SurroGate system greatly enhances the screening efficiency of plant base-edited cells.Mol. Plant. 2020; 13: 169-180Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar).