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作物学报 ›› 2020, Vol. 46 ›› Issue (7): 978-986.doi: 10.3724/SP.J.1006.2020.93064

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于CRISPR/Cas9核糖核蛋白体DNA定点内切酶体外活性建立高效基因型分析技术

王南1,祁显涛1,2,刘昌林1,谢传晓1,*(),朱金洁1,*()   

  1. 1 中国农业科学院作物科学研究所, 北京100081
    2 安徽农业大学, 安徽合肥230036
  • 收稿日期:2019-12-18 接受日期:2020-03-24 出版日期:2020-07-12 网络出版日期:2020-04-10
  • 通讯作者: 谢传晓,朱金洁
  • 作者简介:E-mail: 2720409346@qq.com
  • 基金资助:
    国家转基因生物新品种培育重大专项(2019ZX08010-003)

Establishment of an efficient genotyping technique based on targeted DNA endonuclease in vitro activity of CRISPR/Cas9 ribonucleoprotein

WANG Nan1,QI Xian-Tao1,2,LIU Chang-Lin1,XIE Chuan-Xiao1,*(),ZHU Jin-Jie1,*()   

  1. 1 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    2 Anhui Agricultural University, Hefei 230036, Anhui, China
  • Received:2019-12-18 Accepted:2020-03-24 Published:2020-07-12 Published online:2020-04-10
  • Contact: Chuan-Xiao XIE,Jin-Jie ZHU
  • Supported by:
    National Major Project of Developing New GM Crops(2019ZX08010-003)

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摘要：

建立快速、准确、高通量与简便易行的基因型分析技术对基因功能解析、分子育种与突变体鉴定研究具有重要价值。本研究的目标是利用Cas9或Cas9NG变体与单分子指导RNA (single guide RNA, sgRNA)核糖核蛋白复合体(sgRNA/Cas9-RNP或sgRNA/Cas9NG-RNP)体外DNA定点内切酶活性, 建立与优化简便高效与低成本的基因型分析技术。以我们前期创制的CRISPR/Cas9定点编辑玉米ZmWx基因第7外显子区域定点突变基因编辑后代分离群体为材料, 以ZmWx靶位点两侧特异引物扩增的PCR产物为底物, 利用原核表达并纯化的Cas9或Cas9-NG蛋白为DNA内切酶, 以体外转录的靶向ZmWx基因靶点的sgRNA或骨架序列优化的sgRNA (enhanced sgRNA, esgRNA)为Cas9或Cas9-NG酶定点活性指导元件, 通过体外组装为sgRNA/Cas9-RNP复合体, 对目标样本进行酶切, 以区分目标位点野生型、纯合突变体、杂合突变体基因型, 并对反应体系进行了优化。研究表明, 基于esgRNA/Cas9的PCR/RNP检测技术可对ZmWx基因编辑目标突变体后代进行快速有效的基因型鉴定; 实验体系优化结果表明, esgRNA/Cas9蛋白质量比为1:1, 各为1 μg, 20 μL反应体系, 37℃酶切0.5 h, 可对500 ng待测DNA底物充分酶切并确定基因型; esgRNA/Cas9NG反应体系优化结果表明, 当esgRNA与Cas9-NG蛋白均为2 μg时, 37℃酶切4 h, 可对500 ng DNA底物进行酶切并实现基因型分析。利用Cas9NG拓宽靶位点检测范围的研究结果, 暗示Cas9NG是以牺牲核酸酶酶切活性为代价降低了Cas9蛋白对PAM (protospacer adjacent motif, PAM)基序NGG序列的依赖性, 实现PAM-NG基序识别能力, sgRNA/Cas9NG检测效率等仍有待提升与优化。本研究为基因功能解析、分子育种与突变体鉴定等研究提供了一套简便、成本低廉的技术方法, Cas9NG体外内切酶活性及其效率也为该Cas9突变体活体基因编辑技术研发提供了参考数据。

关键词: sgRNA/Cas9核糖核蛋白复合体, Cas9NG, 优化sgRNA (esgRNA), 基因型分析, 突变体筛选

Abstract:

Establishing a rapid, accurate, high-throughput and easily implementable genotyping method is highly desirable for functional genomics, genetic improvement and mutant screening. Here, we describe a convenient and inexpensive technique for genotyping using the targeted DNA endonuclease activity of Cas9 or Cas9NG ribonucleoproteins complex (sgRNA/Cas9-RNP or sgRNA/Cas9NG-RNP). In this study, Cas9 and Cas9NG protein purified from E. coli extract was assembled with in vitro transcribed single guide RNA (sgRNA)or enhanced sgRNA (esgRNA) as an assembled ribonucleoprotein (RNP) complex to fulfill the targeted endonuclease activity on the PCR amplicons of ZmWx exon 7. The restriction profiles can be converted into genotyping results of wildtype, homozygous or heterozygous mutant, respectively. Our data showed that ZmWx gene-edited mutants can be genotyped rapidly and efficiently by the sgRNA-optimized system esgRNA/Cas9. The reaction component optimization data suggested that 500 ng of DNA substrates could be cleavaged completely by incubating with 1 μg 1:1 molar ratio of esgRNA/Cas9 ribonucleoproteins for 30 minutes at 37℃, or by 4 μg 1:1 molar ratio of esgRNA/Cas9NG ribonucleoproteins for 4 hours at 37℃. Expanding the targeting flexibility of mutant detection via esgRNA/Cas9NG indicated that Cas9NG variant might recognize relax NG PAM (protospacer-adjacent-motif, PAM) at the expense of decreasing restriction activity, which is necessary to improve the activity of esgRNA/Cas9NG by further optimization. Therefore, the establishment and application of esgRNA/Cas9 based PCR/RNP technique provides an easy, simple and low-cost approach to genotyping in functional genomics, molecular breeding and mutant screening. In addition, our in vitro data on esgRNA/Cas9NG has certain and significant reference value for developing it into in vivo genome editing studies.

Key words: sgRNA/Cas9 ribonucleoprotein complex, Cas9NG, enhanced single guide RNA (esgRNA), genotyping, mutant screening

表1

本研究所用引物"

引物名称
Primer name		 序列
Sequence (5′-3′)
Cas9 F GACGACGACGACAAGGCCATGATGGACAAGAAGTACTCCA
Cas9 R TCGACGGAGCTCGCTAGCGTCGCCGCCGAGCTGGGAGAGG
Cas9NG F ACGACGACGACAAGGCCATGGACAAGAAGTACTCCATCGGC
Cas9NG R CTCGAGTGCGGCCGCAAGCTTGTCGCCGCCGAGCTGGCTCAG
sgRNA scaffold F GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG
sgRNA scaffold R AAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAA
esgRNA scaffold F GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATC
esgRNA scaffold R AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTC
ZmWx F CATACTTCTCCGGACCATACGGTAA
ZmWx R TCCCTGCTGGGGTCCCACTC
T7 F AAGCTAATACGACTCACTATAGGAGGTTCAGCTCCGGGTAGTGTTTTAGAGCTAGAAA
eT7 F AAGCTAATACGACTCACTATAGGAGGTTCAGCTCCGGGTAGTGTTTCAGAGCTATGCT
Target1 T7 F AAGCTAATACGACTCACTATAGGTTCAGCTCCGGGTAGTCGGGTTTCAGAGCTATGCT
Target2 T7 F AAGCTAATACGACTCACTATAGCTCCGGGTAGTCGGAGAAGGGTTTCAGAGCTATGCT
Target3 T7 F AAGCTAATACGACTCACTATAGCTTCTCCGACTACCCGGAGCGTTTCAGAGCTATGCT
Target4 T7 F AAGCTAATACGACTCACTATAGTTCGCCTTCTCCGACTACCCGTTTCAGAGCTATGCT
Target5 T7 F AAGCTAATACGACTCACTATAGGCCTTCTCCGACTACCCGGAGTTTCAGAGCTATGCT
Target6 T7 F AAGCTAATACGACTCACTATAGCAGCTCCGGGTAGTCGGAGAGTTTCAGAGCTATGCT
T7 R AAAAAGCACCGACTCGGTGCCACTTTTT

图1

Cas9与Cas9NG蛋白的原核表达及纯化 A: 原核表达载体结构示意图; B: Cas9纯化蛋白10% SDS-PAGE胶图; C: Cas9-NG纯化蛋白10% SDS-PAGE胶图。M: Protein marker; 1: 上清液中蛋白; 2: Ni柱纯化蛋白; 3: 阳离子交换柱纯化蛋白。"

图2

sgRNA、esgRNA体外转录及基于esgRNA/Cas9-RNP和esgRNA/Cas9NG-RNP检测体系的建立 A: sgRNA二级结构图; B: esgRNA二级结构图; C: RNA的10% Urea-PAGE胶图; D: PCR/RNP酶切检测; CK: 未加esgRNA和Cas蛋白的ZmWx野生型PCR产物。"

图3

基于esgRNA /Cas9-RNP及esgRNA/ Cas9NG-RNP检测体系的优化 A: esgRNA梯度;B: Cas9蛋白梯度; C: 酶切反应时间梯度; D: 不同基因型PCR产物酶切; E: esgRNA梯度; F: Cas9NG蛋白梯度; G: 酶切反应时间梯度; H: 不同基因型PCR产物酶切; CK: 未加esgRNA和Cas蛋白的ZmWx 野生型PCR产物。"

图4

基于esgRNA/Cas9NG-RNP检测体系对不同ZmWx位点的检测 A: Cas9-NG的6个新的靶向位点选择; B: 6个靶点对应的esgRNA 10% Urea-PAGE鉴定; C: PCR/RNP酶切分析; CK: 未加esgRNA和Cas蛋白的ZmWx野生型PCR产物。"

图5

基于esgRNA/Cas9-RNP的ZmWx基因编辑突变体检测 A: 32个ZmWx的基因编辑材料的esgRNA/Cas9-RNP酶切检测; B: Sanger测序峰图。WT/WT为野生型, -1 bp/-1 bp为缺失1 bp的纯合突变体, WT/-3 bp为一个等位基因缺失3 bp的杂合突变体; CK: 未加esgRNA和Cas蛋白的ZmWx野生型PCR产物。"

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