Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.
Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.
复制标题
DOI：
10.1038/s41467-022-28135-9
复制
发表时间：
2022-01-21
影响因子：
16.6
通讯作者：
Xue W
中科院分区：
文献类型：
--
作者：
Liang SQ;Liu P;Smith JL;Mintzer E;Maitland S;Dong X;Yang Q;Lee J;Haynes CM;Zhu LJ;Watts JK;Sontheimer EJ;Wolfe SA;Xue W
脱靶编辑的分析是开发安全的基于核酸酶的基因组编辑疗法的重要方面。核酸酶脱靶活性的体内评估主要是间接的（基于体外、细胞中的发现或通过计算预测）或通过基于ChIP的双链断裂（DSB）DNA修复因子的检测，这可能很麻烦。在本文中，我们描述了GUIDE-tag，其能够在小鼠肝脏和肺中进行一步脱靶基因组编辑分析。GUIDE标签系统利用Cas9核酸酶和DNA供体之间的拴系来增加核酸酶介导的DSB的捕获率，并通过Tn 5标签片段化来增加UMI掺入以避免PCR偏倚。这些组分可以作为SpyCas 9-mSA核糖核蛋白复合物和生物素-dsDNA供体递送，用于体内编辑分析。GUIDE-tag能够检测编辑率≥ 0.2%的脱靶位点。利用相同的标签片段化基因组DNA的UDiTaS分析检测体内具有脱靶位点和大缺失的低频易位事件。SpyCas 9-mSA和生物素-dsDNA系统提供了一种在多种组织中体内捕获DSB基因座的方法，其工作流程适合于分析与基因组编辑相关的总体基因组改变。核酸酶脱靶活性的体内评估主要是间接的或通过双链断裂DNA修复因子的基于ChIP的检测，这可能很麻烦。在这里，作者表明GUIDE-tag能够在小鼠肝脏和肺部进行一步脱靶基因组编辑分析。
Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing. In vivo assessment of nuclease off-target activity has primarily been indirect or through ChIP-based detection of double-strand break DNA repair factors, which can be cumbersome. Here, the authors show that GUIDE-tag, enables one-step off-target genome editing analysis in mouse liver and lung.
登录
查看更多内容
影响因子：
7
作者：
Auer TO;Duroure K;De Cian A;Concordet JP;Del Bene F
通讯作者：
Del Bene F
影响因子：
16.6
作者：
Bolukbasi MF;Liu P;Luk K;Kwok SF;Gupta A;Amrani N;Sontheimer EJ;Zhu LJ;Wolfe SA
通讯作者：
Wolfe SA
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
14.9
作者：
He X;Tan C;Wang F;Wang Y;Zhou R;Cui D;You W;Zhao H;Ren J;Feng B
通讯作者：
Feng B