生物技术通报 ›› 2021, Vol. 37 ›› Issue (6): 279-285.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0231

• 技术与方法 • 上一篇    下一篇

水稻CRISPR/Cas12a系统的优化及其介导的腺嘌呤碱基编辑器的建立

王敬文1(), 严芳1, 柳浪1, 周雪平1,2, 王道文3,4, 周焕斌1,5()   

  1. 1.中国农业科学院植物保护研究所 植物病虫害生物学国家重点实验室,北京 100193
    2.浙江大学农业与生物技术学院,杭州 310058
    3.中国科学院遗传与发育生物学研究所 植物细胞与染色体工程国家重点实验室,北京 100101
    4.河南农业大学农学院,郑州 450002
    5.农业农村部桂林作物有害生物科学观测实验站,桂林 541399
  • 收稿日期:2020-03-01 出版日期:2021-06-26 发布日期:2021-07-08
  • 作者简介:王敬文,硕士研究生,研究方向:基因组编缉技术开发与应用;E-mail: wangjingwenHN@163.com
  • 基金资助:
    国家自然科学基金项目(31871948);中国农业科学院科技创新工程(Y2020PT26)

Optimization of CRISPR/Cas12a System and Development of It-mediated Adenine Base Editor in Rice

WANG Jing-wen1(), YAN Fang1, LIU Lang1, ZHOU Xue-ping1,2, WANG Dao-wen3,4, ZHOU Huan-bin1,5()   

  1. 1. State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193
    2. College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058
    3. State Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101
    4. College of Agronomy,Henan Agricultural University,Zhengzhou 450002
    5. Scientific Observing and Experimental Station of Crop Pests in Guilin,Ministry of Agriculture and Rural Affairs,Guilin 541399
  • Received:2020-03-01 Published:2021-06-26 Online:2021-07-08

摘要:

为了构建高效的CRISPR/Cas12a介导的水稻基因编辑系统,通过测试不同来源的Cas12a蛋白、crRNA长度和构型来探索影响Cas12a编辑活性的因素发现,相对于AsCas12a和LbCas12a在crRNA为23 nt时具有更高的编辑效率,并且crRNA 3'端的U4AU4结构并不能增强其在水稻中的编辑活性,同时利用大肠杆菌腺嘌呤脱氨酶变体TadA8e开发了CRISPR/LbCas12a介导的腺嘌呤碱基编辑系统pUbi∶rBE58,成功地将水稻内源基因OsCPK15实现A到G替换(A>G),其效率为33.33%。本研究证明了利用CRISPR/Cas12a可以对水稻基因组进行编辑,丰富了水稻基因编辑工具,拓宽了基因编辑工具箱。

关键词: CRISPR, Cas12a, 腺嘌呤碱基编辑器, 水稻

Abstract:

In order to develop an efficient CRISPR/Cas12a-mediated gene editing system in rice,different Cas12a homologous proteins and crRNA configurations and lengths were tested to explore the factors affecting the editing activity of Cas12a. We found that LbCas12a had a high editing efficiency with crRNA 23 nt compared with AsCas12a,and the U4AU4 sequence at the 3' end of crRNA didn’t enhanced its editing activity. Meanwhile,we fused the Escherichia coli adenine deaminase variant TadA8e to develop the CRISPR/LbCas12a-mediated adenine base editing system pUbi∶rBE58,which successfully induced the conversion A to G at the target loci in rice endogenous gene OsCPK15,with an efficiency of 33.33%. Our results demonstrate that using CRISPR/Cas12a-mediated may genetically edit the genes of rice,and this enriches rice gene editing tools and broadens gene editing toolkits.

Key words: CRISPR, Cas12a, adenine base editor, Oryza sativa