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作物学报 ›› 2019, Vol. 45 ›› Issue (6): 848-855.doi: 10.3724/SP.J.1006.2019.82052

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

一个CRISPR/Cas9-VQR基因编辑系统的构建

陈凯,孙国梁,宋高原,李爱丽,谢传晓,毛龙(),耿帅锋()   

  1. 中国农业科学院作物科学研究所, 北京 100081
  • 收稿日期:2018-10-24 接受日期:2019-01-19 出版日期:2019-06-12 网络出版日期:2019-06-12
  • 通讯作者: 陈凯,孙国梁,毛龙,耿帅锋
  • 作者简介:陈凯, E-mail: 575961814@qq.com|孙国梁, E-mail: sunglCAAS@126.com
  • 基金资助:
    本研究由国家转基因生物新品种培育重大专项(2016ZX080009-001)

Establishment of a CRISPR/Cas9-VQR gene editing system

Kai CHEN,Guo-Liang SUN,Gao-Yuan SONG,Ai-Li LI,Chuan-Xiao XIE,Long MAO(),Shuai-Feng GENG()   

  1. Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2018-10-24 Accepted:2019-01-19 Published:2019-06-12 Published online:2019-06-12
  • Contact: Kai CHEN,Guo-Liang SUN,Long MAO,Shuai-Feng GENG
  • Supported by:
    This study was supported by the National Major Project for Developing New GM Crops(2016ZX080009-001)

摘要:

CRISPR/Cas9系统是一种广泛应用于细菌、酵母、动物和植物中的基因组定点编辑技术, 但该编辑系统的使用范围受PAM (proto-spacer-motif)位点NGG的限制。本研究通过突变Streptococcus pyogenes Cas9 (SpCas9)编码氨基酸 (1135位的天冬氨酸D突变成缬氨酸V, 1335位的精氨酸R突变为谷胱氨酸Q, 1337位的苏氨酸T突变为精氨酸R, 命名该突变子为Cas9-VQR)改造其识别PAM为NGA的位点以扩大其使用范围。并使用玉米Ubi启动子启动Cas9-VQR基因、优化SpCas9的密码子、加入保守的核定位信号序列、增加单子叶植物中保守的 3′ UTR 序列和使用水稻U6启动子启动gRNA来修饰该编辑系统。结果表明Cas9-VQR系统能够识别PAM为NGA的位点, 并进行有效的切割。体外酶切活性检测结果表明Cas9-VQR的切割效率为5%~70%。水稻转化检测结果表明Cas9-VQR的切割效率约为27.5%~70.5%, 平均切割效率为46.23%。本研究拓宽了CRISPR/Cas9系统在作物中的使用范围, 特别是NGA PAM位点较高的作物。

关键词: CRISPR/Cas9-VQR, 定点突变, PAM, 基因突变

Abstract:

Clustered Regularly Interspaced Short Palindromic Repeat and Cas9 (CRISPR/Cas9), a new generation of genome- editing technology, is widely applied among bacteria, yeast, animals and plants, however, the typical CRISRP/Cas9 cannot recognize the NGA proto-spacer-motif (PAM), which limits its application. In order to broaden the applications of CRIPSR/Cas9 system, we modified the Streptococcus pyogenes Cas9 (SpCas9) sequence by the PCR site-direct mutagenesis, which encodes V (1135), Q (1335), and R (1337), to make the CRIPSR/Cas9-VQR able to recognize the NGA PAM motif. We also constructed a binary expression vector of CRISRP/Cas9-VQR with maize ubiquitin as the promoter to drive the Cas9-VQR, optimizing SpCas9-codon, adding conserved nuclear localization signal sequence, and increasing the conserved 3' UTR sequence of monocots, and using OsU6 transcripts of sRNA. CRISPR/Cas9-VQR could recognize the NGA motif and cut targeted sequence in vivo. We assembled the Cas9-VQR protein with the sRNAs in vitro. The Cas9-VQR could cleave the targeted fragments with about 5%-70% of mutation efficiency. In the transformation of rice, we detected about 27.50%-70.50% of mutation ratio, with an average of 46.23%. This system broadens the CRISPR/Cas9 applications in crops, especially in these with higher PAM locus of NGA.

Key words: CRISPR/Cas9-VQR, site-direct mutagenesis, PAM, mutation rate

表1

OsPDS-gRNAs序列"

靶位点
Target site
序列
Targeted sequence (5′-3′)
GC含量
GC content (%)
外显子位置
Location
OsPDS-gRNA1 CTTGGAAGGATGAAGATGGAGA 45 3
OsPDS-gRNA2 CCAGGAGAATTCAGCCGGTTTGA 55 6
OsPDS-gRNA3 TCAGGAGAAGCATGGTTCTAAGA 45 8
OsPDS-gRNA4 TCCCGGACTGTGAACCTTGCCGA 60 13

表2

本研究所用引物"

引物名称
Primer name
序列
Sequence (5′-3′)
T7-gRNA1-FPg TAATACGACTCACTATAGCTTGGAAGGATGAAGATGGGTTTTAGAGCTAGAAATAGC
T7-gRNA2-FPg TAATACGACTCACTATAGCCAGGAGAATTCAGCCGGTTGTTTTAGAGCTAGAAATAGC
T7-gRNA3-FPg TAATACGACTCACTATAGTCAGGAGAAGCATGGTTCTAGTTTTAGAGCTAGAAATAGC
T7-gRNA4-FPg TAATACGACTCACTATAGTCCCGGACTGTGAACCTTGCGTTTTAGAGCTAGAAATAGC
gRNA-RP AGCACCGACTCGGTGCCACTT
oligo-gRNA1-F TTGCTTGGAAGGATGAAGATGG
oligo-gRNA1-R AACCCATCTTCATCCTTCCAAG
oligo-gRNA2-F TTGCCAGGAGAATTCAGCCGGTT
oligo-gRNA2-R AACAACCGGCTGAATTCTCCTGG
oligo-gRNA4-F TTGTCCCGGACTGTGAACCTTGC
oligo-gRNA4-R AACGCAAGGTTCACAGTCCGGGA
PCR-gRNA1-F TCAGAGTAAAGCAAAGATTC
PCR-gRNA1-R TCAGGCTCCTACGAGATA
PCR-gRNA2-F GAATTTTCGCTTAGAGGC
PCR-gRNA2-R CCAGTTATTTGAGTTCCATC
PCR-gRNA4-F TGTGCCTGTATGTAACCA
PCR-gRNA4-R GAGCAAACTTCACCTTCT

表3

Cas9密码子优化结果"

名称
Name
长度
Length
(bp)
G含量
G contents
C含量
C contents
GC百分比
GC percent (%)
SpCas9 4116 733 566 31.56
Cas9-codon optimized 4116 1094 931 49.20

图1

水稻和小麦全基因组CDS中PAM位点的数量"

图2

CRISPR/Cas9-VQR载体 ZmUbi: 玉米泛素启动子; NLS: 核定位信号; D1135V: 1135位的天冬氨酸(D)突变为缬氨酸(V); R1335Q: 1335位的精氨酸(R)突变为谷胱氨酸(Q); T1337R: 1337位的苏氨酸(T)突变为精氨酸(R); 3′UTR: 3′非翻译区; Ter: 终止子; gRNA: 指导RNA; OsU6: 水稻U6启动子。"

图3

体外酶切检测活性结果 S1和S2: 标准样品1和2; CK1和CK2: 阴性对照1和2; g1: OsPDS-gRNA1; g2: OsPDS-gRNA2; g3: OsPDS-gRNA3; g4: OsPDS-gRNA4。"

图4

OsPDS-gRNA体外活性检测结果"

图5

表型观察 A: 苗期; B: 叶片; C: 穗。WT: 野生型; OsPDS-gRNAs: gRNA载体OsPDS-gRNA1、OsPDS-gRNA2和OsPDS-gRNA4。"

图6

靶位点突变类型 A: OsPDS-gRNA1靶位点的突变类型; B: OsPDS-gRNA2靶位点的突变类型; C: OsPDS-gRNA4靶位点的突变类型。WT: 野生型; L: 不同株系。"

表5

转基因植株突变体数量统计"

名称
Name
阳性植株
Transplant
突变植株
Mutated plant
突变率
Mutation ratio (%)
纯合突变体
Homo plant
杂合突变体
Het plant
OsPDS-gRNA1 17/24 12 70.5 4/12 8/12
OsPDS-gRNA2 40/45 11 27.5 5/11 6/11
OsPDS-gRNA4 49/53 20 40.8 7/20 13/20
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