CRISPR/Cpf1单碱基编辑系统的构建及应用

doi:10.19586/j.2095-2341.2021.0088

生物技术进展 ›› 2021, Vol. 11 ›› Issue (6): 732-740.DOI: 10.19586/j.2095-2341.2021.0088

CRISPR/Cpf1单碱基编辑系统的构建及应用

李树磊(), 徐妙云, 郑红艳(), 王磊()

中国农业科学院生物技术研究所，北京 100081

收稿日期:2021-05-21 接受日期:2021-06-07 出版日期:2021-11-25 发布日期:2021-11-26
通讯作者: 郑红艳,王磊
作者简介:李树磊 E-mail：lishuleilsl@163.com；
基金资助:
三亚崖州湾科技城管理局2020年度科技计划项目(SKJC-2020-02-005)

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

Shulei LI(), Miaoyun XU, Hongyan ZHENG(), Lei WANG()

Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2021-05-21 Accepted:2021-06-07 Online:2021-11-25 Published:2021-11-26
Contact: Hongyan ZHENG,Lei WANG

摘要/Abstract

摘要：

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。

关键词: 单碱基编辑, 玉米, FnCpf1

Abstract:

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.

Key words: base editing, maize, FnCpf1

中图分类号:

李树磊, 徐妙云, 郑红艳, 王磊. CRISPR/Cpf1单碱基编辑系统的构建及应用[J]. 生物技术进展, 2021, 11(6): 732-740.

Shulei LI, Miaoyun XU, Hongyan ZHENG, Lei WANG. Establishment and Application of CRISPR/Cpf1‑mediated Base Editing System[J]. Current Biotechnology, 2021, 11(6): 732-740.

图/表 7

参考文献 24

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引物名称	正向序列（5′→3′）	引物用途
check‑r2‑fw①	5′‑GAACTTGCCCCGTCTTTTGAAT‑3′	巢式PCR第一轮扩增
middle‑R‑rv	5′‑GCAAGTGAATGGTGGTGTCT‑3′	巢式PCR第一轮扩增
NGS‑R2‑F	5′‑CCTTGACAAAGAAGCGTGCC‑3′	巢式PCR第二轮扩增/NGS测序样品制备
NGS‑R2‑R1	5′‑CCAGTTTGGATTATCTGGGC‑3′	巢式PCR第二轮扩增/NGS测序样品制备
FnCpf1 for PB_LP	5′‑AGCGCTACGCCTGAACTTAAGATGTCCATCTACCAGGAGTTCGTG‑3′	扩增dFnCpf1序列
FnCpf1 for PB_RP	5′‑GCTGCCGCCGGAGTCACGCGTGTTGTTCCTGTTCTGCACAAACT‑3′	扩增dFnCpf1序列
PB_cexu	5′‑ACCACAGCTGACCTTCTTCACC‑3′	dFnCpf1元件测序
U6_gRNA_LP	5′‑ACGACGGCCAGTGCCAAGCTTGGATCATGAACCAACGGCCT‑3′	扩增U6+crRNA+spacer 序列
U6_gRNA_bt2_RP2	5′‑GCACTGCAGGCATGCAAGCTTAAAAAAAAGCAACATATCCAAGATCTATGTGCATCTACAACAGTAGAAATTA‑3′	扩增U6+crRNA+spacer 序列，引入R2 spacer序列
R2‑OTA‑F	5′‑GAAACTCCATCACATTATGC‑3′	扩增R2‑OT‑A脱靶位点
R2‑OTA‑R	5′‑AGCAGAAGGATTGTCCCCTC‑3′	扩增R2‑OT‑A脱靶位点
R2‑OTB‑F	5′‑CCTTGCCGTCAGCTTATTAGAT‑3′	扩增R2‑OT‑B脱靶位点
R2‑OTB‑R	5′‑TCCTGTTATCAGCATGAACT‑3′	扩增R2‑OT‑B脱靶位点
R2‑OTC‑F	5′‑GGCGAAGGTGACGAAGAGT‑3′	扩增R2‑OT‑C 脱靶位点
R2‑OTC‑R	5′‑TACAACTCTAGCACACGCCTC‑3′	扩增R2‑OT‑C 脱靶位点

引物名称	正向序列（5′→3′）	引物用途
check‑r2‑fw①	5′‑GAACTTGCCCCGTCTTTTGAAT‑3′	巢式PCR第一轮扩增
middle‑R‑rv	5′‑GCAAGTGAATGGTGGTGTCT‑3′	巢式PCR第一轮扩增
NGS‑R2‑F	5′‑CCTTGACAAAGAAGCGTGCC‑3′	巢式PCR第二轮扩增/NGS测序样品制备
NGS‑R2‑R1	5′‑CCAGTTTGGATTATCTGGGC‑3′	巢式PCR第二轮扩增/NGS测序样品制备
FnCpf1 for PB_LP	5′‑AGCGCTACGCCTGAACTTAAGATGTCCATCTACCAGGAGTTCGTG‑3′	扩增dFnCpf1序列
FnCpf1 for PB_RP	5′‑GCTGCCGCCGGAGTCACGCGTGTTGTTCCTGTTCTGCACAAACT‑3′	扩增dFnCpf1序列
PB_cexu	5′‑ACCACAGCTGACCTTCTTCACC‑3′	dFnCpf1元件测序
U6_gRNA_LP	5′‑ACGACGGCCAGTGCCAAGCTTGGATCATGAACCAACGGCCT‑3′	扩增U6+crRNA+spacer 序列
U6_gRNA_bt2_RP2	5′‑GCACTGCAGGCATGCAAGCTTAAAAAAAAGCAACATATCCAAGATCTATGTGCATCTACAACAGTAGAAATTA‑3′	扩增U6+crRNA+spacer 序列，引入R2 spacer序列
R2‑OTA‑F	5′‑GAAACTCCATCACATTATGC‑3′	扩增R2‑OT‑A脱靶位点
R2‑OTA‑R	5′‑AGCAGAAGGATTGTCCCCTC‑3′	扩增R2‑OT‑A脱靶位点
R2‑OTB‑F	5′‑CCTTGCCGTCAGCTTATTAGAT‑3′	扩增R2‑OT‑B脱靶位点
R2‑OTB‑R	5′‑TCCTGTTATCAGCATGAACT‑3′	扩增R2‑OT‑B脱靶位点
R2‑OTC‑F	5′‑GGCGAAGGTGACGAAGAGT‑3′	扩增R2‑OT‑C 脱靶位点
R2‑OTC‑R	5′‑TACAACTCTAGCACACGCCTC‑3′	扩增R2‑OT‑C 脱靶位点

靶位点	基因组定位	碱基错配个数	靶位点序列（5′→3′）
R2‑OT‑A	1:61546022‑61546048	1	5′‑TTAGCACATAGATCTTGGATATGTTGT‑3′
R2‑OT‑B	2:178205267‑178205293	4	5′‑TTAGCACATATATCTTGGAGACGTTAC‑3′
R2‑OT‑C	1:11161065‑11161091	5	5′‑TTTACACATATATCTTGGGTTTGCTGC‑3′

靶位点	基因组定位	碱基错配个数	靶位点序列（5′→3′）
R2‑OT‑A	1:61546022‑61546048	1	5′‑TTAGCACATAGATCTTGGATATGTTGT‑3′
R2‑OT‑B	2:178205267‑178205293	4	5′‑TTAGCACATATATCTTGGAGACGTTAC‑3′
R2‑OT‑C	1:11161065‑11161091	5	5′‑TTTACACATATATCTTGGGTTTGCTGC‑3′

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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

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