Highly efficient base editing with expanded targeting scope using SpCas9-NG in rabbits

被引:21
作者
Liu, Zhiquan [1 ]
Shan, Huanhuan [1 ]
Chen, Siyu [1 ]
Chen, Mao [1 ]
Song, Yuning [1 ]
Lai, Liangxue [1 ,2 ,3 ,4 ]
Li, Zhanjun [1 ]
机构
[1] Jilin Univ, Coll Anim Sci, Key Lab Zoonosis Res, Minist Educ, Changchun 130062, Peoples R China
[2] Chinese Acad Sci, Guangdong Prov Key Lab Stem Cell & Regenerat Med, South China Inst Stem Cell Biol & Regenerat Med, CAS Key Lab Regenerat Biol,Guangzhou Inst Biomed, Guangzhou, Peoples R China
[3] GRMH GDL, Guangzhou, Peoples R China
[4] Chinese Acad Sci, Inst Stem Cell & Regenerat, Beijing, Peoples R China
关键词
base editor; CRISPR; Cas9; NG PAMs; rabbit; GENOMIC DNA; HAIR; MUTATION; FGF5; IDENTIFICATION; DELETION; LONG;
D O I
10.1096/fj.201901587R
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Base editors, composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable efficient C-to-T or A-to-G conversion in various organisms. However, the NGG protospacer adjacent motif (PAM) requirement of Streptococcus pyogenes Cas9 (SpCas9) substantially limits the target sites suitable for base editing. Quite recently, a new engineered SpCas9-NG variant, which can recognize minimal NG PAMs more efficiently than the present xCas9 variant. Here, we investigated the efficiency and PAM compatibility of SpCas9-NG-assisted cytidine base editors (CBEs) and adenine base editors (ABEs) in rabbits. In this study, we showed that NG-BE4max and NG-ABEmax systems can achieve a targeted mutation efficiency of 75%-100% and 80%-100% with excellent PAM compatibility of NGN PAMs in rabbit embryos, respectively. In addition, both base editors were successfully applied to create new rabbit models with precise point mutations, demonstrating their high efficiency and expanded genome-targeting scope in rabbits. Meanwhile, NG-ABEmax can be used to precisely mimic human Hoxc13 p.Q271R missense mutation in Founder (F0) rabbits, which is arduous for conventional ABEs to achieve due to a NGA PAM requirement. Collectively, NG-BE4max and NG-ABEmax systems provide promising tools to perform efficient base editing with expanded targeting scope in rabbits and enhances its capacity to model human diseases.
引用
收藏
页码:588 / 596
页数:9
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