识别NG PAM序列的Cas9变异体编辑玉米ZmSCD基因及其效率测试  

Identification and Efficiency Testing of Cas9 Variants Targeting NG PAM Sequences for Editing the ZmSCD Gene in Maize

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作  者:钱步轩 潘弘 王棋 陈子奇 杨雅文 许洁婷 夏涵超 赵仁贵[1] 刘相国 QIAN Buxuan;PAN Hong;WANG Qi;CHEN Ziqi;YANG Yawen;XU Jieting;XIA Hanchao;ZHAO Rengui;LIU Xiangguo(Faculty of Agronomy,Jilin Agricultural University,Changchun 130033,China;Institute of Agricultural Biotechnology(Jilin Provincial Key Laboratory of Agricultural Biotechnology),Jilin Academy of Agricultural Sciences(Northeast Agricultural Research Center of China),Changchun 130033,China;WIMI Biotechnology Co.,Ltd,Changzhou 213000,China)

机构地区:[1]吉林农业大学农学院,吉林长春130033 [2]吉林省农业科学院(中国农业科技东北创新中心),农业生物技术研究所(吉林省农业生物技术重点实验室),吉林长春130033 [3]未米生物科技(江苏)有限公司,江苏常州213000

出  处:《华北农学报》2024年第5期9-14,共6页Acta Agriculturae Boreali-Sinica

基  金:吉林省农业科技创新工程(CXGC2024RCG002);吉林省农业科技创新工程人才基金(CXGC2022RCY009);国家自然科学基金面上项目(32171928)。

摘  要:为了探究评估SpCas9-NG在玉米基因组编辑中的应用潜力,以叶绿素合成关键基因ZmSCD作为编辑对象,该基因缺失会使苗出现白化现象,以此直观评估编辑效率。依据SpCas9和SpCas9-NG分别识别的5′-NGG-3′和5′-NG-3′PAM序列的规则,在ZmSCD的第2和第3外显子上设计了靶点后,成功将靶点序列构建至SpCas9及SpCas9-NG敲除载体上,再利用农杆菌介导的遗传转化方法将敲除载体导入玉米自交系KN5585中,将愈伤组织培养至分化出叶片组织时,统计白化苗率,以此得出不同编辑器的编辑效率,并对白化苗提取基因组后进行测序。通过3轮遗传转化SpCas9分别获得76,125,28个愈伤组织,SpCas9-NG获得100,69,30个愈伤组织。结果显示,SpCas9的3轮遗传转化的基因编辑效率分别为14.47%,13.60%,10.71%,SpCas9-NG编辑效率分别为12.00%,10.14%,13.33%;对其白化苗测序结果显示,均在靶点附近获得重叠峰。结果表明,SpCas9-NG在玉米中的编辑效率与传统SpCas9相似,显示出同样的基因编辑能力;相比之下,SpCas9-NG具备更宽泛的PAM序列适应性,这意味着它的靶点设计能力更为灵活,允许在玉米基因组中实现更加精准和多样化的编辑。To evaluate the potential of SpCas9-NG in maize genome editing,the key gene for chlorophyll synthesis,ZmSCD,was selected as the target.The absence of this gene causes seedlings to exhibit albinism,providing a visual assessment of editing efficiency.Based on the PAM sequence recognition rules of SpCas9(5′-NGG-3′)and SpCas9-NG(5′-NG-3′),target sites were designed on the second and third exons of ZmSCD.These target sequences were successfully constructed into SpCas9 and SpCas9-NG knockout vectors,which were then introduced into the maize inbred line KN5585 using Agrobacterium-mediated genetic transformation.The callus tissues were cultured until leaf tissues differentiated,and the albinism rate was recorded to determine the editing efficiency of the different editors.The genomes of albino seedlings were then sequenced.Through three rounds of genetic transformation,SpCas9 produced 76,125 and 28 callus tissues,while SpCas9-NG produced 100,69 and 30 callus tissues.The results showed that the gene editing efficiency of SpCas9 across the three transformations was 14.47%,13.60%,and 10.71%,respectively,while SpCas9-NG editing efficiency was 12.00%,10.14%and 13.33%.Sequencing results of the albino seedlings revealed overlapping peaks near the target sites for both editors.The study demonstrates that SpCas9-NG had comparable editing efficiency to traditional SpCas9 in maize,showing similar gene editing capabilities.In contrast,SpCas9-NG has a broader PAM sequence adaptability,allowing for more flexible target design.This flexibility enables more precise and diverse editing within the maize genome.

关 键 词:玉米 SpCas9-NG 编辑效率 ZmSCD 

分 类 号:S513[农业科学—作物学]

 

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