CRISPR/Cpf1单碱基编辑系统的构建及应用

Establishment and Application of CRISPR/Cpf1‑mediated Base Editing System

作物的优良性状往往来自于其相应基因的单个碱基突变,而传统育种无法轻易获得此种定向单碱基变异。单碱基编辑技术是以成簇规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats/CRISPR⁃associated proteins,CRISPR/Cas)系统为基础改良的一项基因编辑技术,该技术可在不造成DNA双链断裂的情况下对靶序列上的特定碱基进行定向替换。为拓展单碱基编辑技术在作物中的识别范围,利用来自Francisella novicida细菌的FnCpf1核酸酶及胞嘧啶脱氨酶APOBEC1对单碱基编辑系统进行改良,并针对玉米BT2基因靶位点构建相应载体,通过瞬时转化手段检测其编辑能力。检测结果发现9种碱基变化类型,其中靶位点5'端第11个碱基的胞嘧啶转化为腺嘌呤,位点编辑效率达到2.5%。结果表明该系统能够识别“TTN”作为原型间隔序列毗邻基序(protospacer⁃adjacent motif,PAM)并对靶位点进行单碱基编辑,为单碱基编辑识别范围的拓展提供了研究思路。

The excellent traits of crops often come from single base mutations in their corresponding genes,and traditional breeding cannot easily obtain such targeted base mutations.The base editing technology is a gene editing technology based on the clustered regularly interspaced short palindromic repeats/CRISPR⁃associated proteins(CRISPR/Cas)system,which can perform targeted replacement of specific bases on the target sequence without causing DNA double⁃strand break.In order to expand the recognition range of base editing technology in crops,the FnCpf1 from Francisella novicida and the cytosine deaminase APOBEC1 were used to improve the base editing system.Then,constructing the corresponding vector targeting to the maize BT2 gene.Detected its editing ability by means of instantaneous conversion.The test results found 9 types of base changes.Among them,the cytosine of the 11th base at the 5'end of the target site was converted to adenine,and the base editing efficiency reached 2.5%.It showed that the system can recognize"TTN"as a protospacer⁃adjacent motif(PAM)site and single⁃base editing of the target site,which can provide research ideas for the expansion of the recognition range of base editing.