基于CRISPR/CasX介导的水稻基因组编辑技术的建立  

Establishment of CRISPR/CasX-based Genome Editing Technology in Rice

作　　者：李雪琪 张素杰 于曼 黄金光 周焕斌 LI Xue-qi;ZHANG Su-jie;YU Man;HUANG Jin-guang;ZHOU Huan-bin(College of Plant Health and Medicine,Qingdao Agricultural University,Qingdao 266109;State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100093;Scientific Observing and Experimental Station of Crop Pests in Guilin,Ministry of Agriculture and Rural Affairs,Guilin 541399)

机构地区：[1]青岛农业大学植物医学学院,青岛266109 [2]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,北京100093 [3]农业农村部桂林作物有害生物科学观测实验站,桂林541399

出　　处：《生物技术通报》2023年第9期40-48,共9页Biotechnology Bulletin

基　　金：海南省崖州湾种子实验室资助项目(B21HJ0215);农业农村部基因编辑创新利用重点实验室(海南);中国农业科学院农业科技创新工程。

摘　　要：Cas蛋白作为核酸酶发挥其切割活性需要识别特定的PAM序列,如SpCas9识别NGG PAM位点,LbCas12a识别TTTV PAM。已挖掘到新的能够识别TTCN PAM序列的蛋白—CasX蛋白,扩展了基因组编辑技术的编辑范围。本研究利用CasX的两个衍生型蛋白PlmCasX和DpbCasX,建立基于CRISPR/CasX介导的水稻基因编辑系统。通过PEG介导的水稻原生质体瞬时表达分析其编辑活性发现,PlmCasX和DpbCasX两个蛋白能够对水稻内源基因OsCPK16实现有效编辑。后通过水稻稳定遗传转化进一步验证,在TTCA PAM识别位点,DpbCasX蛋白对水稻内源基因OsCPK21的编辑效率为17.5%,PlmCasX蛋白对水稻内源基因OsCPK21的编辑效率为66.07%;在TTCG PAM识别位点,PlmCasX蛋白对OsCPK4的编辑效率为23.21%,而DpbCasX蛋白不能实现有效的基因编辑。并且基于MIDAS方法对PlmCasX蛋白的优化并不能提高其编辑活性。本研究证明了CRISPR/CasX系统在水稻中具有编辑活性,且其能识别TTCR PAM这一特性,扩大了基因编辑技术在水稻中的应用范围。As a nuclease,the Cas protein needs to recognize a specific protospacer adjacent motifs(PAM)sequence to exert its cleavage activity,for example SpCas9 recognizes the NGG PAM and LbCas12a recognizes the TTTV PAM.CasX protein,a new protein that can recognize the TTCN PAM sequence was discovered,which expands the editing range of genome editing technology.Using PlmCasX and DpbCasX derived from CasX,CRISPR/CasX-mediated rice gene editing system was established.The editing efficiency through transient expression of rice protoplasts mediated with PEG was analysed,and the results showed that the endogenous OsCPK16 was effectively edited by PlmCasX and DpbCasX.The subsequent rice stable genetic transformation results indicated DpbCasX showed 17.5%editing efficiency in OsCPK21 at TTCA PAM sites,while PlmCasX showed higher editing efficiency in OsCPK21 at TTCA PAM sites and in OsCPK4 at TTCG PAM sites,with editing efficiency of 66.07%and 23.21%,respectively.And the optimization of PlmCasX protein based on the MIDAS method could not improve its editing activity.The study proved that the CRISPR/CasX system has editing activity in rice,and it can recognize the characteristic of TTCR PAM,expanding the application range of gene editing technology in rice.

关 键 词：水稻 基因组编辑 CRISPR CasX 

分 类 号：Q78[生物学—分子生物学] S511[农业科学—作物学]