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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2021, Vol. 41 Issue (12): 4-12    DOI: 10.13523/j.cb.2111010
玉米生物育种基础研究与关键技术专辑     
CRISPR/Cas植物基因组编辑技术及其在玉米中的应用*
杨梦冰1,2,江易林1,2,祝蕾1,2,安学丽1,2,3,**(),万向元1,2,3,**()
1 北京科技大学生物与农业研究中心 化学与生物工程学院 顺德研究生院 北京 100083
2 北京中智生物农业国际研究院 北京 100192
3 北京首佳利华科技有限公司 主要作物生物育种北京市工程实验室 生物育种北京市国际科技合作基地 北京 100192
CRISPR/Cas Plant Genome Editing Systems and Their Applications in Maize
YANG Meng-bing1,2,JIANG Yi-lin1,2,ZHU Lei1,2,AN Xue-li1,2,3,**(),WAN Xiang-yuan1,2,3,**()
1 Research Center of Biology and Agriculture, Shunde Graduate School, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China
2 Zhongzhi International Institute of Agricultural Biosciences, Beijing 100192, China
3 Beijing Engineering Laboratory of Main Crop Bio-Tech Breeding, Beijing International Science and Technology Cooperation Base of Bio-Tech Breeding, Beijing Solidwill Sci-Tech Co., Ltd., Beijing 100192, China
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摘要：

CRISPR/Cas 系统具有操作简单、效率高等优势,为植物功能基因研究和作物遗传改良提供了重要支撑。介绍了CRISPR/Cas植物基因组编辑技术的研究进展,并对CRISPR/Cas系统及其衍生技术进行了详细比较;结合案例综述了CRISPR/Cas9基因编辑技术在玉米产量、品质、抗逆性改良,以及雄性不育系创制和单倍体诱导等方面的应用;同时针对CRISPR/Cas系统未来需要迫切解决的一些问题进行了分析和展望。
关键词： CRISPR/Cas系统;  基因组编辑;  植物基因组;  玉米遗传改良    
Abstract:

Due to their simplicity and high efficiency, CRISPR/Cas systems provide efficient tools for functional genomics and crop molecular breeding. In this review, we summarize the current developments of CRISPR/Cas genomic editing systems in plants and compare the differences between these systems and their derivative technologies. We review the applications of CRISPR/Cas9 editing technology in maize improvement focusing on yield, quality, disease resistance, abiotic stress resistance, male sterile line development and haploid induction. Finally, we discuss the future improvement of CRISPR/Cas systems and provide perspectives on prospect genome editing technologies.
Key words: CRISPR/Cas systems    Genome editing    Plant genome    Genetic improvement of maize
收稿日期: 2021-11-02 出版日期: 2022-01-13
ZTFLH:  Q819  
基金资助: * 国家自然科学基金面上项目(31971958);中央高校基本科研业务费专项资金(06500060)
通讯作者: 安学丽,万向元     E-mail: xuelian@ustb.edu.cn;wanxiangyuan@ustb.edu.cn
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杨梦冰
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万向元

引用本文:

杨梦冰,江易林,祝蕾,安学丽,万向元. CRISPR/Cas植物基因组编辑技术及其在玉米中的应用*[J]. 中国生物工程杂志, 2021, 41(12): 4-12.

YANG Meng-bing,JIANG Yi-lin,ZHU Lei,AN Xue-li,WAN Xiang-yuan. CRISPR/Cas Plant Genome Editing Systems and Their Applications in Maize. China Biotechnology, 2021, 41(12): 4-12.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2111010    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I12/4

比较项目 CRISPR/Cas9 CRISPR/Cas12 CRISPR/Cas13 单碱基编辑 引导编辑
分类 Class II Type II Class II Type V Class II Type VI CRISPR/Cas9衍生工具 CRISPR/Cas9衍生工具
核心元件 tracRNA、crRNA、
Cas9		 crRNA、Cas12a或
Cas12b		 crRNA、Cas13a、
Cas13b或Cas13d		 胞嘧啶或腺嘌呤脱氨酶、UGI、nCas9、sgRNA nCas9、M-MLV逆转录酶、pegRNA
关键结构域 RuvC、HNH RuvC-like、 Nuc HEPN HNH RuvC
靶向核酸类型 dsDNA dsDNA ssRNA dsDNA dsDNA
靶位点 PAM序列的5'端2~3 nt处 PAM序列下游18 nt处 和23 nt处 靶标ssRNA,可非特异性切割ssRNA PAM序列上游的C或A(5~12碱基内) 单链缺口处
靶位点限制 PAM序列通常为NGG 严格的 PAM 序列“TTTV” 需识别或不需识别PFS序列 PAM序列通常为NGG 受PAM限制较小
编辑效率 高 较高 较高 较高 低
脱靶频率 较高 低 较高 低 较低
优势 载体构建简单,可实现多靶点编辑 适用于更多装载量小的载体系统,有利于DNA片段的定点插入和替换 可以特异性靶向切割病毒RNA和真核细胞中的内源RNA 可以实现精确的碱基替换 有效地产生精确的碱基替换、插入和缺失等突变
缺点 会对细胞基因组造成一定的损伤 低温下编辑效率低 具有非特异性切割活性,脱靶率较高 同一编辑系统对不同靶点的编辑效率差异较大 在植物中的编辑效率是极低和不稳定
表1  CRISPR/Cas系统及衍生技术的比较
图1  CRISPR/Cas9和CRISPR/Cas12a基因编辑系统的工作原理
图2  基于CRISPR/Cas9系统的玉米基因组编辑技术流程
应用 靶标基因 基因功能 编辑方式 性状改良 参考文献
提高产量 ZmLG1 编码SBP转录因子类蛋白 碱基插入和缺失 减小叶夹角,提高种植密度 [30]
ZmCLE7 控制分生组织大小 碱基插入和缺失 行和籽粒产量增加 [31]
ZmFCP1
改善品质 ZmWx1 编码GBSS酶 碱基插入和缺失 糯玉米 [32]
ZmSh2 编码AGPase酶 碱基插入和缺失 甜玉米 [33]
增强抗逆性 ARGOS8 乙烯反应的负调节因子 片段插入 提高耐旱能力 [34]
ZmALS2 编码乙酰乳酸合成酶 碱基插入和缺失 抗除草剂 [35]
ZmALS1 编码乙酰乳酸合成酶 碱基替换 抗除草剂 [36]
ZmALS2
stiff1 编码F-box结构域蛋白 碱基缺失 提高茎秆强度,抗倒伏 [37]
GA20ox3 赤霉素生物合成相关基因 碱基插入和缺失 降低株高,抗倒伏 [38]
LOX3 编码脂氧合酶 碱基插入和缺失 抗玉米黑粉菌感染 [39]
调控开花时间 ZmPHYC1 编码玉米光敏色素蛋白 碱基插入和缺失 长日照条件表现为早花 [40]
ZmPHYC2
ZmCCT9 编码CCT转录因子 碱基缺失 长日照条件引起早开花 [41]
创制雄性不育系 MS26 编码细胞色素P450 碱基插入和缺失 花粉发育异常,雄性不育 [35]
MS45 编码异胡豆苷合成酶类似蛋白 碱基插入和缺失 花粉发育异常,雄性不育 [35]
ZmTMS5 编码RNase Z蛋白 碱基缺失 温敏雄性不育植株 [42]
ZmMS8 编码β-1,3-半乳糖基转移酶 碱基插入和缺失 花药发育异常,雄性不育 [43]
Dcl5 介导产生24 nt phasiRNA 碱基插入和缺失 温敏雄性不育植株 [44]
单倍体育种 ZmDMP 编码DUF679膜蛋白 碱基插入和缺失 诱导单倍体产生 [45]
ZmPLA1 编码磷脂酶A 碱基插入和缺失 诱导单倍体产生 [46]
多性状改良 - - 片段插入 多性状基因的聚合 [47]
表2  CRISPR/Cas9技术在玉米改良中的应用
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