Abstract:
Base editors that do not require double-stranded DNA cleavage or homology-directed repair enable higher efficiency and cleaner substitution of targeted single nucleotides in genomic DNA than conventional approaches. However, their broad applications are limited within the editing window of several base pairs from the canonical NGG protospacer adjacent motif (PAM) sequence. In this study, we fused the D10A nickase of several Streptococcus pyogenes Cas9 (SpCas9) variants with Petromyzon marinus cytidine deaminase 1 (PmCDA1) and uracil DNA glycosylase inhibitor (UGI) and developed two new effective PmCDA1-based cytosine base editors (pBEs), SpCas9 nickase (SpCas9n)-pBE and VQR nickase (VQRn)-pBE, which expanded the scope of genome targeting for cytosine-to-thymine (C-to-T) substitutions in rice. Four of six and 12 of 18 target sites selected randomly in SpCas9n-pBE and VQRn-pBE, respectively were base edited with frequencies of 4-90% in T0 plants. The effective deaminase window typically spanned positions 1-7 within the protospacer and the single target C showed the maximum C-to-T frequency at or near position 3, counting the end distal to PAM as position 1. In addition, the modified single guide RNA (sgRNA) improved the base editing efficiencies of VQRn-pBE with 1.3- to 7.6-fold increases compared with the native sgRNA, and targets that could not be mutated using the native sgRNA were edited successfully using the modified sgRNA. These newly developed base editors can be used to realize C-to-T substitutions and may become powerful tools for both basic scientific research and crop breeding in rice.
摘要:
与常规方法相比,不需要双链DNA切割或同源定向修复的碱基编辑器能够实现基因组DNA中靶向单核苷酸的更高效率和更清洁的取代。然而,它们的广泛应用限于来自规范NGG原型间隔区相邻基序(PAM)序列的几个碱基对的编辑窗口内。在这项研究中,我们融合了几种化脓性链球菌Cas9(SpCas9)变体的D10A切口酶与Petromyzonmarinus胞苷脱氨酶1(PmCDA1)和尿嘧啶DNA糖基化酶抑制剂(UGI),并开发了两种新的有效的基于PmCDA1的胞嘧啶碱基编辑器(pBE),SpCas9切口酶(SpCas9n)-pBE和VQR切口酶(VQRn)-pBE,扩大了水稻中胞嘧啶到胸腺嘧啶(C到T)替换的基因组靶向范围。在SpCas9n-pBE和VQRn-pBE中随机选择的18个靶位点中的6个和12个,分别在T0植物中以4-90%的频率进行碱基编辑。有效的脱氨酶窗口通常跨越原型间隔物内的位置1-7,单个靶标C在位置3或附近显示出最大的C到T频率,将PAM的远端计数为位置1。此外,与天然sgRNA相比,修饰的单向导RNA(sgRNA)提高了VQRn-pBE的碱基编辑效率,增加了1.3至7.6倍,和不能使用天然sgRNA突变的靶标使用修饰的sgRNA成功地编辑。这些新开发的基础编辑器可用于实现C到T替换,并可能成为水稻基础科学研究和作物育种的强大工具。