Highly efficient base editing in rabbit by using near-PAMless engineered CRISPR/Cas9 variants
Highly efficient base editing in rabbit by using near-PAMless engineered CRISPR/Cas9 variants
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DOI：
10.1007/s11427-021-2165-1
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发表时间：
2022-09
期刊：
影响因子：
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通讯作者：
Zhongtian Zhang;Xinyu Wu;Jie Yang;Xin Liu;Ruonan Liu;Yuning Song
中科院分区：
文献类型：
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作者：
Zhongtian Zhang;Xinyu Wu;Jie Yang;Xin Liu;Ruonan Liu;Yuning Song
碱基编辑可以实现有针对性的C-T和A-G转换，而不需要产生DNA双链断裂(DSB)或需要供体模板，从而显示出产生新突变或纠正致病突变的潜力(Gaudelli等人，2018年)。然而，对于传统的碱基编辑来说，有效的碱基编辑需要存在位于目标碱基下游12-16个核苷酸的NGG Protspacer相邻基序(PAM)，这通常限制了这些碱基编辑的应用(Gaudelli等人，2018年)。为了提高碱基编辑系统的通用性，引入了各种修改以提高PAM的兼容性、编辑效率和特定主题的专一性。例如，eA3G-BE可以识别NG PAM的基因座(Liu等人，2020b)，YFE-BE4max具有更窄的编辑窗口和更低的旁观者活性(Liu等人，2020a)，而Nme2-CBE具有N4CC PAM偏好(Liu等人，2021)。然而，仍然有一些非常规PAM(如NCN、NAN)的位置不能被基础编辑瞄准。因此，基础编辑系统需要进一步扩展PAM兼容性。最近，据报道，两个名为SPG(NGN PAMS)和SPRY(NRN，在较小程度上，NYN PAM)的CRISPR/Cas9工程变体在保持高编辑效率的同时扩展了PAM兼容性(Walton等人，2020)。在此，我们旨在探讨无PAM的SPG和SPRY系统在兔体内的可行性和有效性。为了评估SPG-BE4max在兔胚胎中的编辑活性，选择了三个带有NGN PAM的靶基因(Tyr、DMD和MSTN)用于SPG-BE4max介导的C-T转换(图S1a在支持信息中)。在兔胚胎中进行碱基编辑，使用编码BE的mRNA和单引导RNA的联合显微注射。如支持信息中的图S1B-D所示，SPG-BE4max的C-to-T编辑效率在40%到100%之间，高于SpCas9-NG系统，SpCas9-NG系统是另一种SpCas9变体系统，在以前的报告(任等，2019年)中显示出对人类细胞中NG PAM的高效，这表明它在兔胚胎中含有NG PAM的基因座上具有出色的编辑效率。此外，还在SOD1、Lmna和TDP43基因的座位上测试了SpGABEmax和NG-ABEmax介导的A-G碱基替换的效率(支持信息中的图S2A)。这些结果表明，在兔胚胎中，SPG-ABEmax在含有NG PAM的座位上的效率高于SpCas9-NG系统(支持信息中的图S2B-D)。
Base editors can achieve targeted C-to-T and A-to-G conversion without the generation of DNA double-strand breaks (DSBs) or the requirement of a donor template, showing the potential to generate new mutations or to correct pathogenic mutations (Gaudelli et al., 2018). However, for conventional base editors, efficient base editing requires the presence of an NGG protospacer adjacent motif (PAM) that lies 12–16 nt downstream of the target bases, which generally limits the application of these base editors (Gaudelli et al., 2018). To improve the versatility of base editing systems, various modifications have been introduced to promote PAM compatibility, editing efficiency and specificity in specific motifs. For instance, eA3G-BE can recognize loci with NG PAM (Liu et al., 2020b), YFE-BE4max has a narrowed editing window and reduced bystander activity (Liu et al., 2020a), and Nme2-CBE has an N4CC PAM preference (Liu et al., 2021). However, there are still loci with unconventional PAMs (eg, NCN, NAN) that cannot be targeted by base editors. Therefore, further expansion of PAM compatibility is needed for base editing systems. Recently, two engineered CRISPR/Cas9 variants, named SpG (NGN PAMs) and SpRY (NRN and, to a lesser extent, NYN PAMs), were reported to have expanded PAM compatibility with maintained high editing efficiency (Walton et al., 2020). Here, we aimed to explore the feasibility and efficacy ofPAM-less SpG and SpRY systems in rabbits. To assess the editing activity of SpG-BE4max in rabbit embryos, three target loci with NGN PAMs (Tyr, Dmd, and Mstn) were selected for SpG-BE4max-mediated C-to-T conversion (Figure S1A in Supporting Information). Base editing was conducted in rabbit embryos using co-microinjection of BE-encoding mRNA and single-guide RNAs. As shown in Figure S1B–D in Supporting Information, the C-to-T editing efficiency of SpG-BE4max ranges from 40% to 100%, which is higher than that of the SpCas9-NG system, another SpCas9 variant system that showed high efficiency toward NG PAMs in human cells in previous reports (Ren et al., 2019), suggesting its outstanding editing efficiency at loci containing NG PAMs in rabbit embryos. Moreover, the efficiency of A-to-G base substitution mediated by SpGABEmax and NG-ABEmax was also tested in loci from the Sod1, Lmna and Tdp43 genes (Figure S2A in Supporting Information). These results indicate that SpG-ABEmax has higher efficiency at loci with NG PAMs than the SpCas9-NG system in rabbit embryos (Figure S2B–D in Supporting Information).