qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites.
qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites.
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DOI：
10.1093/nar/gky505
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发表时间：
2018-09-28
影响因子：
14.9
通讯作者：
Kozlowski P
中科院分区：
文献类型：
--
作者：
Dabrowska M;Czubak K;Juzwa W;Krzyzosiak WJ;Olejniczak M;Kozlowski P
基于工程化核酸酶的基因组编辑技术已经越来越多地应用于多种细胞类型和生物体中的基因的靶向修饰。然而，目前用于评估编辑效率的方法仍然受到许多限制，包括优先检测某些突变类型、对阻碍错配检测的多态性敏感、缺乏多重能力或对测定条件敏感。在这里，我们描述了qEva-CRISPR，这是一种新的定量方法，它克服了这些限制，并允许同时（多重）分析CRISPR/Cas9诱导的靶点和相应的脱靶点或几个不同靶点的修饰。我们在不同的细胞系和不同的实验条件下使用靶向TP 53、VEGFA、CCR 5、EMX 1和HTT基因的许多sgRNA证明了qEva-CRISPR方法的所有优点。与其他方法不同，qEva-CRISPR检测所有类型的突变，包括点突变和大缺失，其灵敏度不依赖于突变类型。此外，这种方法允许成功分析位于“困难”基因组区域的靶标。总之，qEva-CRISPR可能成为无偏sgRNA筛选的首选方法，以评估影响基因组编辑的实验条件或区分同源性定向修复与非同源末端连接。
Genome editing technology based on engineered nucleases has been increasingly applied for targeted modification of genes in a variety of cell types and organisms. However, the methods currently used for evaluating the editing efficiency still suffer from many limitations, including preferential detection of some mutation types, sensitivity to polymorphisms that hamper mismatch detection, lack of multiplex capability, or sensitivity to assay conditions. Here, we describe qEva-CRISPR, a new quantitative method that overcomes these limitations and allows simultaneous (multiplex) analysis of CRISPR/Cas9-induced modifications in a target and the corresponding off-targets or in several different targets. We demonstrate all of the advantages of the qEva-CRISPR method using a number of sgRNAs targeting the TP53, VEGFA, CCR5, EMX1 and HTT genes in different cell lines and under different experimental conditions. Unlike other methods, qEva-CRISPR detects all types of mutations, including point mutations and large deletions, and its sensitivity does not depend on the mutation type. Moreover, this approach allows for successful analysis of targets located in ‘difficult’ genomic regions. In conclusion, qEva-CRISPR may become a method of choice for unbiased sgRNA screening to evaluate experimental conditions that affect genome editing or to distinguish homology-directed repair from non-homologous end joining.
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影响因子：
14.9
作者：
Brinkman, Eva K.;Kousholt, Arne N.;van Steensel, Bas
通讯作者：
van Steensel, Bas
影响因子：
7
作者：
Cho SW;Kim S;Kim Y;Kweon J;Kim HS;Bae S;Kim JS
通讯作者：
Kim JS
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
影响因子：
3.7
作者：
Lewandowska MA;Czubak K;Klonowska K;Jozwicki W;Kowalewski J;Kozlowski P
通讯作者：
Kozlowski P
影响因子：
--
作者：
Marcinkowska, Malgorzata;Wong, Kwok-Kin;Kozlowski, Piotr
通讯作者：
Kozlowski, Piotr