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Research Articles

Cas9 protein variant VQR recognizes NGAC protospacer adjacent motif in rice

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  • 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 date: 2018-05-10

  Revised date: 2018-06-20

  Online published: 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)

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.

Cite this article

Gaowei Xin, Xixun Hu, Kejian Wang, Xingchun Wang . Cas9 protein variant VQR recognizes NGAC protospacer adjacent motif in rice[J]. Hereditas(Beijing), 2018 , 40(12) : 1112 -1119 . DOI: 10.16288/j.yczz.18-126

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