Expanding the targeting scope of CRISPR/Cas9-mediated genome editing by Cas9 variants in Brassica

作     者：Wenjing Li Xuan Li Chunyang Wang Guanzhong Huo Xinru Zhang Jintai Yu Xiaoxiao Yu Jing Li Chao Zhang Jianjun Zhao Yan Li Jun Li 

作者机构：State Key Laboratory of North China Crop Improvement and RegulationCollege of Life SciencesHebei Agricultural UniversityBaoding 071001China Hebei Key Laboratory of Plant Physiology and Molecular PathologyHebei Agricultural UniversityBaoding 071001China Key Laboratory of Vegetable Germplasm Innovation and Utilization of HebeiCollaborative Innovation Center of Vegetable Industry in HebeiCollege of HorticultureHebei Agricultural UniversityBaoding 071001China College of Modern Science and TechnologyHebei Agricultural UniversityBaoding 071001China 

出 版 物：《aBIOTECH》 (生物技术通报（英文版）)

年 卷 期：2024年第5卷第2期

页      面：202-208页

核心收录：

学科分类：0710[理学-生物学] 07[理学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 090102[农学-作物遗传育种] 

基　　金：supported by the S&T Program of Hebei(21372901D 23567601H) Key project of National Natural Science Foundation of China(32330096) the Natural Science Foundation of Hebei(C2023204119) the Starting Grant from Hebei Agricultural University(YJ201958) 

主　　题：Brassica Cas9 variants CRISPR Genome editing Non-canonical PAM 

摘      要：CRISPR/Cas9,presently the most widely used genome editing technology,has provided great potential for functional studies and plant ***,the strict requirement for a protospacer adjacent motif(PAM)has hindered the application of the CRISPR/Cas9 system because the number of targetable genomic sites is ***,the engineered variants Cas9-NG,SpG,and SpRY,which recognize non-canonical PAMs,have been successfully tested in plants(mainly in rice,a monocot).In this study,we evaluated the targeted mutagenesis capabilities of these Cas9 variants in two important Brassica vegetables,Chinese cabbage(Brassica rapa ***)and cabbage(Brassica oleracea ***).Both Cas9-NG and SpG induced efficient mutagenesis at NGN PAMs,while SpG outperformed Cas9-NG at NGC and NGT *** achieved efficient editing at almost all PAMs(NRNNYN),albeit with some self-targeting activity at transfer(T)-DNA *** SpRY-induced mutants were detected in cabbage plants in a PAM-less ***,an adenine base editor was developed using SpRY and TadA8e deaminase that induced A-to-G conversions within target sites using non-canonical ***,the toolboxes developed here induced successful genome editing in Chinese cabbage and *** work further expands the targeting scope of genome editing and paves the way for future basic research and genetic improvement in Brassica.