基于CRISPR/LanCas9的水稻基因编辑系统的开发和优化  

Development and Optimization of Genome Editing in Rice with CRISPR/LanCas9 System

作　　者：李欣格 王美霞 王晨阳[2] 马桂根 周常勇[3] 王亚南[1] 周焕斌[2,4,5] LI Xin-ge;WANG Mei-xia;WANG Chen-yang;MA Gui-gen;ZHOU Chang-yong;WANG Ya-nan;ZHOU Huan-bin(College of Plant Protection,Hebei Agricultural University,Baoding 071001;State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193;National Citrus Engineering Research Center,Citrus Research Institute,Southwest University,Chongqing 400712;Key Laboratory of Gene Editing Technologies(Hainan),Ministry of Agricultural and Rural Affairs,Sanya 572024;Scientific Observing and Experimental Station of Crop Pests in Guilin,Ministry of Agriculture and Rural Affairs,Guilin 541399)

机构地区：[1]河北农业大学植物保护学院,保定071001 [2]中国农业科学院植物保护所植物病虫害综合治理全国重点实验室,北京100193 [3]西南大学柑橘研究所国家柑橘工程研究中心,重庆400712 [4]农业农村部基因编辑创新利用重点实验室(海南),三亚572024 [5]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》2024年第10期233-242,共10页Biotechnology Bulletin

基　　金：科技创新2030—重大项目(2023ZD0407405-03)。

摘　　要：【目的】在水稻中建立CRISPR/LanCas9和CRISPR/SLanCas9基因编辑系统,扩展CRISPR/Cas基因编辑工具箱。【方法】将来自动物乳酸杆菌KCTC 3501的LanCas9进行密码子优化,并且进一步通过蛋白融合重组LanCas9和SpCas9的活性结构域形成嵌合体SLanCas9,分别构建CRISPR/LanCas9和CRISPR/SLanCas9编辑工具;以OsWRKY45、OsCPK4、OsCPK6、OsCPK7、OsMPK8、OsGSK3、OsGSK4为靶基因,在NGG PAM或者NAG PAM设计20 nt或者24 nt的sgRNA,通过水稻遗传转化,检测分析其编辑效率。【结果】LanCas9在水稻中可以识别NGG PAM,结合20 nt的sgRNA编辑效率更高,对OsWRKY45基因的编辑效率为25.00%;融合的SLanCas9不仅能够识别NGG PAM,在NAG PAM位点也有一定的编辑效率,在不同PAM位点的编辑效率分别可达100%和39.58%。此外,SLanCas9还具有多重基因编辑能力,在NGG PAM位点的多重基因编辑效率高达74.07%。【结论】开发了具有自主知识产权的新型CRISPR/LanCas9和CRISPR/SLanCas9基因编辑技术。【Objective】The objective of this study is to develop CRISPR/LanCas9 and CRISPR/SLanCas9 editing systems for rice and to expand the CRISPR/Cas gene-editing toolkit【.Method】The codon of LanCas9 from Lactobacillus subtilis KCTC 3501 was optimized,and further a chimeric SLanCas9 by fusing the active domains of LanCas9 and SpCas9 was generated.Thus CRISPR/LanCas9 and CRISPR/SLanCas9 editing systems for rice were constructed respectively.Using OsWRKY45,OsCPK4,OsCPK6,OsCPK7,OsMPK8,OsGSK3,and OsGSK4 as target genes,the 20 nt or 24 nt sgRNAs in NGG PAM or NAG PAM were designed,and their editing efficiency was analyzed through rice genetic transformation.【Result】The LanCas9 efficiently identified NGG PAM and efficiency was higher when combined with a 20-nt sgRNA editor,resulting in an editing efficiency of 25.00%for the OsWRKY45 gene.Furthermore,the fused SLanCas9 not only showed the recognition of the NGG PAM and also demonstrated a certain level of editing efficiency at the NAG PAM site,achieving editing efficiencies of 100%and 39.58%respectively for different PAM sites.Additionally,SLanCas9 demonstrated multiple-gene editing capabilities with an impressive efficiency of up to 74.07%at NGG PAM sites【.Conclusion】This study has successfully developed novel CRISPR/LanCas9 and CRISPR/SLanCas9 gene editing technologies with independent intellectual property rights.

关 键 词：CRISPR LanCas9 SLanCas9 基因编辑 水稻 

分 类 号：Q943.2[生物学—植物学] S511[农业科学—作物学]