CRISPR RNA Array‐Guided Multisite Cleavage for Gene Disruption by Cas9 and Cpf1
CRISPR RNA Array‐Guided Multisite Cleavage for Gene Disruption by Cas9 and Cpf1
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DOI：
10.1002/cbic.201800241
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发表时间：
2018-09
期刊：
影响因子：
3.2
通讯作者：
Dan Wang;Dejun Ma;Jingxin Han;Linghao Kong;Lu-Yuan Li;Z. Xi
中科院分区：
文献类型：
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作者：
Dan Wang;Dejun Ma;Jingxin Han;Linghao Kong;Lu-Yuan Li;Z. Xi
为了同时实现基于多位点靶向的DNA切割，我们设计了两种CRISPR RNA阵列，通过将四种单向导RNA（Cas9的sgRNA或Cpf1的crRNA）与不可切割的RNA接头（CRISPRay）融合。CRISPRay可以在四个相邻的靶位点上操作，以协同的方式切割靶DNA。两个CRISPR RNA阵列证明了萤火虫荧光素酶基因在活细胞中的稳健失活。体外DNA切割和DNA测序也证实了sgRNA阵列指导SpCas9核酸酶同时在四个切割位点切割靶DNA，而crRNA阵列指导的FnCpf1核酸酶在随机位点对靶DNA和非靶DNA都显示出靶激活的非特异性DNA酶活性。通过优化核酸酶和CRIPSR RNA的比例，CRISPRay应该进一步增强细胞中的基因干扰。这项工作提出了一种简单的方法，通过将四种指导RNA（sgRNA或crRNA）融合到CRISPR RNA串中来改善多位点定向基因破坏。
To achieve multisite‐targeting‐based DNA cleavage simultaneously, we designed two kinds of CRISPR RNA arrays by fusing four single guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) with uncleavable RNA linkers (CRISPRay). The CRISPRay could operate on four adjacent target sites to cleave target DNA in a collaborative manner. Two CRISPR RNA arrays demonstrated robust inactivation of the firefly luciferase gene in living cells. In vitro DNA cleavage and DNA sequencing also verified that sgRNA arrays directed SpCas9 nuclease to cut target DNA at four cleavage sites simultaneously whereas crRNA‐array‐guided FnCpf1 nuclease showed target‐activated, nonspecific DNase activity on both target DNA and nontarget DNA at random sites. Through optimization of the ratio of nuclease and CRIPSR RNAs, CRISPRay should further enhance gene interference in cells. This work presents a simple approach through which to improve multisite‐directed gene disruption by fusing four guide RNAs (sgRNAs or crRNAs) into a CRISPR RNA string.