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

Base editors (BEs), including cytosine base editor (CBE) and adenine (ABE), have been widely used to generate irreversible nucleotide substitution in plants animals. However, their wide applications are largely hindered by the strict NG protospacer adjacent motif (PAM) sequences recognized Streptococcus pyogenes Cas9 (SpCas9) its engineered variants, such as SpCas9-NG xCas9 (Hua et al., 2019Hua K. Tao X. Han P. Wang R. Zhu J.K. Genome engineering rice using variants that recognize PAM sequences.Mol. Plant. 2019; 12: 1003-1014Abstract Full Text PDF PubMed Scopus (53) Google Scholar; Ren 2019Ren B. Liu L. Li S. Kuang Y. J. Zhang D. Zhou Lin H. Cas9-NG greatly expands targeting scope of genome-editing toolkit recognizing other atypical PAMs rice.Mol. 1015-1026Abstract Wu 2019Wu Xu W. F. Zhao Feng Song C. Yang Increasing editing efficiency with cas9-PmCDA1 fusions modified sgRNA rice.Front. Genet. 10: 379Crossref (22) Zhong 2019Zhong Z. Sretenovic Q. Bao Qi Yuan M. He al.Improving plant genome high-fidelity non-canonical PAM-targeting Cas9-NG.Mol. 1027-1036Abstract (73) 2020Zhang Kang G. Lv Expanding GA relaxed sites improved system.Plant Biotechnol. 2020; 18: 884-886Crossref (9) Scholar). Most recently, it was reported three new SpCas9 SpCas9-NRRH, SpCas9-NRTH, SpCas9-NRCH, could non-G (NRNH, where R is A or G H A, C, T) human cells (Miller 2020Miller S.M. T. Randolph P.B. Arbab Shen M.W. Huang T.P. Matuszek Newby G.A. Rees H.A. D.R. Continuous evolution compatible PAMs.Nat. 38: 471-481Crossref (93) Meanwhile, SpRY, another variant, developed expand BEs nearly PAMless (Walton 2020Walton R.T. Christie K.A. Whittaker M.N. Kleinstiver B.P. Unconstrained near-PAMless CRISPR-Cas9 variants.Science. 368: 290-296Crossref (223) In this study, we generated a series efficient BE toolkits almost achieved C-to-T mutation without restriction except for NTG PAM, expanded A-to-G stable transformed rice, providing reference application plants. Given broad compatibility SpRY good performance cells, speculated be also utilized BEs' rice. We first applied CBE system. The D10A nickase plant-favored codon-optimized (SpRYn) (Supplemental Sequence 1) fused N terminus Petromyzon marinus cytidine deaminase1 (PmCDA1) two copies uracil DNA glycosylase inhibitor (UGI), which connected hygromycin phosphotransferase (HPT) P2A, self-cleaving 2A peptide. This fusion protein driven Ubiquitin promoter (OsUbi). tRNA together enhanced single guide RNA (esgRNA) placed under control U6 promoter. Targets were finally added method described Ma 2015Ma Chen Qiu al.A robust CRISPR/Cas9n system convenient, high-efficiency multiplex monocot dicot plants.Mol. 2015; 8: 1274-1284Abstract (870) Scholar. designated SpRYn-CBE (Figure 1A). Considering activity on NRN (where G) chose 12 NAN from six different genes assessment 1B Supplemental Table 1). T0 plants, displayed NAC NAG efficiencies 62.2%, 13.2%, 38.1%, 45.2%, respectively Figure no lines edited at four NAA (NAA-C1 NAA-C4) NAT (NAT-C1 NAT-C4), unlike Another (NAA-C5 NAA-C8) (NAT-C5 NAT-C8) tested further identification, but none them mutated either Tables 1 2). Since well SpNRRH SpNRTH, Scholar), SpNRRHn-CBE SpNRTHn-CBE test showed superior more than 90% (NAA-C2 moderate 39.1% target NAA-C1 enabled effective frequencies 18.8% 87.5%, Collectively, these results demonstrate (NAC, NAG) (NAA) (NAT) Next, evaluated eight harboring NGN 1C detectable mutations all sites, ranging 4.2% 37.1% 1), meant an genomic just like involved CBE. As possessed lower NGC (Nishimasu 2018Nishimasu Shi Ishiguro Gao Hirano Okazaki Noda Abudayyeh O.O. Gootenberg J.S. Mori al.Engineered nuclease space.Science. 2018; 361: 1259-1262Crossref (381) SpCas9n-NG-CBE 3) compared SpRYn-CBE. Both NGC-C1 NGC-C2, SpRYn-CBE, not (NGC-C3 NGC-C5) then selected comparison induce whereas did conversion NGC-C5 site These suggest outperforms might act alternative Additionally, assessed activities SpCas9n-CBE NGG sites. found comparable For NYN (Y = C PAMs, five NCN NTN across NTH (H showing 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), 15.4% (NTC-C1) detected indicate those PAMs. Taken together, above capable Based CBE, function expanding ABE. SpRYn HPT Escherichia coli adenosine deaminase variant (ecTadA∗) SpRYn-ABE 1D). Sixteen NAN, NGN, 10 NCN, 9 chosen evaluation 3). efficiently edit NAT, NAC, 85% 1E 4). Unexpectedly, still targeted, quite detect any (NAA-A5 NAA-A8) 3 NGV (V achieving 16.7% (NGA-A1), 11.1%–46.7% (NGC-A1 NGC-A3), 4.8%–19.1% (NGG-A1 NGG-A4) 4), NGT Across bearing NCA, NCG, NTT, 10.5%, 9.5%, 29.2%, 26.7%, NCT, NCC, NTA, NTC, failed NAB (B T, G), NCR (R NTK (K T To address limitation system, SpNRRHn-ABE 1D) SpGn-ABE they although worked well. caused difference between ABE systems. addition, SpCas9n-NG-ABE NGC-A2 NGC-A3 all, 9.1% NGC-A1 (46.7%) especially low SpRYn-ABE, SpCas9n-ABE outperformed preferred All 190 101 taken investigate technical parameters respectively. window spanned positions 13 within protospacer, preference C3 > C2 C4 C5 ≫ Cs 1F). 2 14 1G). A4, A5, A6, A7, A8, A14 frequently (A7 A5 ≈ A6 A8 A4 A14), A2, A3, A9, A11 relatively infrequently (less 10%) 1G), Their windows wider CBEmax (3–16) ABEmax (3–10) type, mutants predominant 5). Single produced position highest each Homozygous Indels seldom Although limited analyzed SpNRTHn-CBE, similar double homozygous T-DNA region, GTT located immediately 3′ end both SpRYn-ABE. self-editing capability T-DNA. Among ineffective one out nine 18 targets had 6). 29 only 15 (6/12) (9/17), there direct relationship occurrence seemed likely self-edited among aforementioned consistent Qin 2020Qin Wei self-targets sequence editing.Nat. Plants. 6: 197-201Crossref (17) Scholar, average higher wild-type data occur mutations, evidence influence ability off-target increase. Four high NN testing identified potential NAC-C1, NTC-C1, NGG-C3 on-target 7). Only deletion 17 NCA-C1 Compared frequency summary, NTH, NAG, Together (NAT), It worth mentioning when SpCas9n-CBE. ABE, outperformance SpCas9n-NG-ABE, Therefore, clear will sharply simplify simplified should maize, wheat, crops future. Further work can carried employ orthologs cover remaining combination DisSUGs technology listed herein may improve enhancing screening base-edited (Xu 2020Xu al.Discriminated sgRNAs-based SurroGate enhances cells.Mol. 13: 169-180Abstract (12)