遗传 ›› 2018, Vol. 40 ›› Issue (12): 1112-1119.doi: 10.16288/j.yczz.18-126

• 研究报告 • 上一篇    下一篇

Cas9蛋白变体VQR高效识别水稻NGAC前间区序列邻近基序

辛高伟1,2,胡熙璕2,王克剑2(),王兴春1()   

  1. 1. 山西农业大学生命科学学院,太谷 030801
    2. 中国农业科学院,中国水稻研究所,水稻生物学国家重点实验室,杭州 310006
  • 收稿日期:2018-05-10 修回日期:2018-06-20 出版日期:2018-12-20 发布日期:2018-09-25
  • 作者简介:辛高伟,硕士研究生,专业方向：生物化学与分子生物学。E-mail: 592950985@qq.com
  • 基金资助:
    中国农业科学院科技创新工程和国家自然科学基金项目资助(31600289)

Cas9 protein variant VQR recognizes NGAC protospacer adjacent motif in rice

Gaowei Xin1,2,Xixun Hu2,Kejian Wang2(),Xingchun Wang1()   

  1. 1. College of Life Sciences, Shanxi Agricultural University, Taigu 030801, China
    2. State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, China
  • Received:2018-05-10 Revised:2018-06-20 Published:2018-12-20 Online:2018-09-25
  • Supported by:
    Supported by the Innovation Project of Chinese Academy of Agricultural Sciences and the National Natural Science Foundation of China(31600289)

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摘要：

成簇的规律间隔短回文重复序列及CRISPR相关蛋白(clustered regularly interspaced short palindromic repeats/CRISPR-associated 9, CRISPR/Cas9)系统是近年来发展起来并被广泛应用的第三代基因组编辑工具。但是,该系统的酿脓链球菌Cas9(Streptococcus pyogenes, SpCas9)仅能识别NGG前间区序列邻近基序(protospacer adjacent motif, PAM),极大地限制了基因组编辑的范围。SpCas9变体VQR(D1135V/R1335Q/T1337R)在水稻中可识别NGAA、NGAG和NGAT PAM,但尚不清楚是否能识别NGAC PAM。本研究利用改进后的CRISPR/VQR系统对水稻中3个相对低效的VQR靶位点NAL1-Q1、NAL1-Q2和LPA1-Q进行了编辑,结果表明改进后的CRISPR/VQR系统可以高效编辑这3个靶位点,编辑效率分别为9.75%、43.90%和29.26%。为了明确改进后的CRISPR/VQR系统对NGAC PAM的识别情况,本研究选择水稻叶片宽度调控基因NARROW LEAF 1 (NAL1)中的NAL-C位点和蜡质合成基因GLOSSY1 (GL1)中的GL1-C位点进行基因编辑,并获得57株转基因水稻。靶位点PCR扩增及测序结果表明,NAL1-C和GL1-C靶标位点突变的植株分别为27株和44株,突变率分别为47.36%和77.19%;其中NAL1-C/GL1-C双突变植株为26株,双突变率为45.61%。进一步分析表明,CRISPR/VQR系统造成的突变有4种类型,分别为杂合突变、双等位突变、嵌合体突变和纯合突变,其中以杂合突变和双等位突变为主。这些结果表明,改进的CRISPR/VQR系统可以高效编辑水稻NGAC PAM位点,并产生丰富的突变类型。本研究为水稻及其他植物相关基因NGAC PAM位点的编辑提供了理论依据。

关键词: 基因编辑, CRISPR/Cas9, VQR, NGAC前间区序列邻近基序, 水稻

Abstract:

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is the third-generation genome editing tools that was developed and widely used in recent years. However, Streptococcus pyogenes Cas9 (SpCas9) in this system could only recognize NGG PAM (protospacer adjacent motif), which largely restricts the range of genome editing. The VQR (D1135V/R1335Q/T1337R) variant of SpCas9 could recognize NGAA, NGAG and NGAT PAMs in rice. However, whether VQR variant could recognize NGAC PAM remains unclear. In this study, three low editing efficiency sites of the VQR variant, NAL1-Q1, NAL1-Q2 and LPA1-Q, were selected for genome editing using the improved CRISPR/VQR system. The improved CRISPR/VQR system effectively edited these target sites, and the gene editing efficiency was 9.75%, 43.90% and 29.26% respectively. To ensure the recognition of NGAC PAM by the improved CRISPR/VQR system, two NGAC PAM containing sites (NAL-C and GL1-C) in the NARROW LEAF 1 (NAL1) for leaf length and GLOSSY1 (GL1) genes for wax biosynthesis were selected for genome editing in rice in this study, and 57 transgenic plants were obtained. The PCR amplification and sequencing results showed that 27 plants (47.36%) had mutation in the NAL1-C site, 44 plants (77.19%) had mutation in the GL1 gene, and 26 plants (45.61%) had mutation in the NAL-C and GL1-C sites. Further analysis revealed that there were four types of mutations caused by the CRISPR/VQR system, respectively for the hybrid mutation, biallelic mutation, chimeric mutation and homozygous mutations. Among them, heterozygous mutation and biallelic mutation were dominant changes. These results indicated that the improved CRISPR/VQR system could efficiently edit the NGAC PAM sites of the rice and produce abundant mutant types. This study provides a theoretical basis for NGAC PAM editing in rice and other related plants.

Key words: gene editing, CRISPR/Cas9, VQR, NGAC PAM, rice