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Action mechanisms of CRISPR/Cas system and its application in genetic improvement of crops |
SHU Xinyuan1, YAN Xu1, PU Yehong1, WANG Chao2, PAN Jianwei2* |
1. College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, Zhejiang, China; 2. School of Life Sciences, Lanzhou University, Lanzhou 730000, China |
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Abstract
CRISPR/Cas system is an emerging gene-editing technology, which can knock out multiple genes at multiple specific loci. Compared with other gene editing systems including ZFNs (zinc finger nucleases) and TALENs (transcription activator-like effector nucleases), CRISPR/Cas system possesses distinguishing advantages including higher targeting efficiency, easier to design and operate, and less time and labor, and thereby is widely applied in microbial, animal and plant genome editing researches. The CRISPR/Cas system was initially found in eubacteria and archaebacteria to be an adaptive immune system that is used to degrade exogenous viruses or plasmids. Multiple CRISPR/Cas systems have been so far reported. CRISPR is a DNA fragment with clustered regularly interspaced short palindromic repeats, which interacts with Cas protein and consequently edits the genome. The main action mechanism of CRISPR/Cas system is that matured crRNA (CRISPR-derived RNA) is hybridized with tracrRNA (trans-activating RNA) to form a single guide RNA (sgRNA), and thereby guides Cas endonuclease to cut double-strand DNA within a 20-nt short sequence, which is complementary with crRNA. Such targeted double-strand breaks activate two distinct DNA repair mechanisms including non-homologous end joining (NHEJ) and homology-directed repair (HDR). The NHEJ mechanism is easy to induce deletion or insertion mutation at the cleavage site. Thus, different sgRNAs can be designed for different target sites to perform gene editing at specific sites. This review mainly focuses on the progress and action mechanisms of CRISPR/Cas9 system and its application in a variety of important crops, and finally makes some prospects about low frequency off-target phenomenon in CRISPR/Cas9 operation and its future applications.
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Published: 28 June 2018
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CRISPR/Cas系统的作用原理及其在作物遗传改良中的应用
CRISPR/Cas系统是一种新兴的基因编辑技术,可在多个特定位点实现多基因敲除。与锌指核酸内切酶(zinc finger nucleases, ZFNs)和类转录激活因子效应物核酸酶(transcription activator-like effector nucleases,TALENs)等基因编辑技术相比,CRISPR/Cas 技术具有靶位点高突变率、易设计和操作、省时省力等特点,被广泛应用于微生物、动物和植物等生物体基因组编辑研究。CRISPR/Cas系统最初在真细菌和古细菌中被发现,是用来降解外源病毒或质粒的一类适应性免疫系统,至今已报道过多种CRISPR/Cas系统。CRISPR由一段成簇有规律的间隔短回文重复序列组成,与Cas 蛋白互作从而对基因组进行基因编辑。CRISPR/Cas系统的主要作用原理是成熟的crRNA与反式激活crRNA(tracrRNA)配对形成单向导RNA(single guide RNA, sgRNA),从而引导Cas 蛋白在特定的20 个核苷酸靶位点处切割双链DNA,由此引发2种不同的DNA修复机制——非同源重组(non-homologous end joining, NHEJ)和同源重组(homology-directed repair, HDR),其中NHEJ在断裂位点诱发碱基缺失或插入突变,因此,可针对不同靶位点设计不同sgRNA在特定的位点实现基因编辑。本文主要介绍了CRISPR/Cas系统的研究进展、作用原理及其在多种重要农作物中的应用,最后对CRISPR/Cas9系统在实际操作中出现的低频脱靶现象和未来应用前景进行了展望。
关键词:
CRISPR/Cas9 系统,
基因编辑,
作物遗传改良,
应用
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