Defining genome-wide CRISPR-Cas genome-editing nuclease activity with GUIDE-seq.
Defining genome-wide CRISPR-Cas genome-editing nuclease activity with GUIDE-seq.
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DOI：
10.1038/s41596-021-00626-x
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发表时间：
2021-12
期刊：
影响因子：
14.8
通讯作者：
Tsai, Shengdar Q.
中科院分区：
文献类型：
--
作者：
Malinin, Nikolay L.;Lee, GaHyun;Lazzarotto, Cicera R.;Li, Yichao;Zheng, Zongli;Nguyen, Nhu T.;Liebers, Matthew;Topkar, Ved V.;Iafrate, A. John;Le, Long P.;Aryee, Martin J.;Joung, J. Keith;Tsai, Shengdar Q.
通过测序实现双链断裂的全基因组无偏鉴定（GUIDE-seq）是一种灵敏、无偏、全基因组的方法，用于定义活细胞中基因组编辑核酸酶的活性。GUIDE-seq的原理是将末端保护的双链寡脱氧核苷酸标签有效整合到核酸酶诱导的DNA双链断裂位点，然后扩增含有标签的基因组DNA分子并进行高通量测序。在这里，我们描述了一个详细的GUIDE-seq协议，包括细胞转染，文库制备，测序和生物信息学分析。包括细胞培养在内的整个过程可在9天内完成。一旦分离出标记整合的基因组DNA，文库制备、测序和分析可在3天内完成。结果是一个脱靶位点的全基因组目录，按核酸酶活性排序，由GUIDE-seq读取计数测量。GUIDE-seq是定义全基因组脱靶活性的最敏感的细胞方法之一，已广泛应用于研究和治疗。
Genome-wide unbiased identification of double stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome editing nucleases in living cells. GUIDE-seq is based on the principle of efficient integration of an end-protected double-stranded oligodeoxynucleotide tag into sites of nuclease-induced DNA double stranded breaks, followed by amplification of tag-containing genomic DNA molecules and high-throughput sequencing. Here we describe a detailed GUIDE-seq protocol including cell transfection, library preparation, sequencing, and bioinformatic analysis. The entire protocol including cell culture can be completed in 9 days. Once tag-integrated genomic DNA is isolated, library preparation, sequencing and analysis can be performed in 3 days. The result is a genome-wide catalogue of off-target sites ranked by nuclease activity as measured by GUIDE-seq read counts. GUIDE-seq is one of the most sensitive cell-based methods for defining genome-wide off-target activity and has been broadly adopted for research and therapeutic use.
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影响因子：
48
作者：
Anderson KR;Haeussler M;Watanabe C;Janakiraman V;Lund J;Modrusan Z;Stinson J;Bei Q;Buechler A;Yu C;Thamminana SR;Tam L;Sowick MA;Alcantar T;O'Neil N;Li J;Ta L;Lima L;Roose-Girma M;Rairdan X;Durinck S;Warming S
通讯作者：
Warming S
影响因子：
46.9
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Kleinstiver BP;Tsai SQ;Prew MS;Nguyen NT;Welch MM;Lopez JM;McCaw ZR;Aryee MJ;Joung JK
通讯作者：
Joung JK
影响因子：
46.9
作者：
Tsai, Shengdar Q.;Zheng, Zongli;Nguyen, Nhu T.;Liebers, Matthew;Topkar, Ved V.;Thapar, Vishal;Wyvekens, Nicolas;Khayter, Cyd;Iafrate, A. John;Le, Long P.;Aryee, Martin J.;Joung, J. Keith
通讯作者：
Joung, J. Keith
影响因子：
48
作者：
Crosetto, Nicola;Mitra, Abhishek;Silva, Maria Joao;Bienko, Magda;Dojer, Norbert;Wang, Qi;Karaca, Elif;Chiarle, Roberto;Skrzypczak, Magdalena;Ginalski, Krzysztof;Pasero, Philippe;Rowicka, Maga;Dikic, Ivan
通讯作者：
Dikic, Ivan
影响因子：
46.9
作者：
Hendel A;Bak RO;Clark JT;Kennedy AB;Ryan DE;Roy S;Steinfeld I;Lunstad BD;Kaiser RJ;Wilkens AB;Bacchetta R;Tsalenko A;Dellinger D;Bruhn L;Porteus MH
通讯作者：
Porteus MH