Programmable DNA repair with CRISPRa/i enhanced homology-directed repair efficiency with a single Cas9.
Programmable DNA repair with CRISPRa/i enhanced homology-directed repair efficiency with a single Cas9.
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使用 CRISPRa/i 进行可编程 DNA 修复，通过单个 Cas9 增强同源定向修复效率
DOI：
10.1038/s41421-018-0049-7
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发表时间：
2018
期刊：
影响因子：
33.5
通讯作者：
Han F
中科院分区：
文献类型：
--
作者：
Ye L;Wang C;Hong L;Sun N;Chen D;Chen S;Han F
CRISPR系统已被证明是哺乳动物物种中位点特异性基因组工程的通用工具。在基因编辑过程中，这些RNA引导核酸酶引入DNA双链断裂（DSB），其中非同源DNA末端连接（NHEJ）主导DNA修复途径，限制了同源定向修复（HDR）的效率，HDR是精确基因靶向所必需的替代途径。已经开发了多种方法来增强HDR，包括化合物或RNA干扰介导的NHEJ因子抑制、HDR酶的小分子激活或CRISPR复合物的细胞周期定时递送。然而，这些方法面临着多重挑战，但具有中等或可变的效果。在这里，我们开发了一种新方法，通过CRISPR激活和干扰（CRISPRa/i）对NHEJ和HDR途径进行编程，以显著提高CRISPR介导的基因编辑的HDR效率。NHEJ和HDR途径组分（如CtIP、CDK 1、KU 70、KU 80和LIG 4）的操纵由催化死亡的向导RNA（dgRNA）介导，因此仅依赖于单个催化活性Cas9来执行CRISPRa/i和精确的基因编辑。虽然大多数DNA修复因子或其组合的重编程测试增强HDR效率，同时激活CDK 1和抑制KU 80具有最强的效果，增加HDR速率高达一个数量级。多西环素诱导的DNA修复酶的基于dgRNA的CRISPRa/i编程以及病毒包装使得能够灵活且可调的HDR增强，以在哺乳动物细胞中具有更广泛的适用性。我们的研究提供了一种有效、灵活且可能更安全的策略来增强精确的基因组修饰，这可能会广泛影响人类基因编辑和治疗。
CRISPR systems have been proven as versatile tools for site-specific genome engineering in mammalian species. During the gene editing processes, these RNA-guide nucleases introduce DNA double strand breaks (DSBs), in which non-homologous DNA end joining (NHEJ) dominates the DNA repair pathway, limiting the efficiency of homology-directed repair (HDR), the alternative pathway essential for precise gene targeting. Multiple approaches have been developed to enhance HDR, including chemical compound or RNA interference-mediated inhibition of NHEJ factors, small molecule activation of HDR enzymes, or cell cycle timed delivery of CRISPR complex. However, these approaches face multiple challenges, yet have moderate or variable effects. Here we developed a new approach that programs both NHEJ and HDR pathways with CRISPR activation and interference (CRISPRa/i) to achieve significantly enhanced HDR efficiency of CRISPR-mediated gene editing. The manipulation of NHEJ and HDR pathway components, such as CtIP, CDK1, KU70, KU80, and LIG4, was mediated by catalytically dead guide RNAs (dgRNAs), thus relying on only a single catalytically active Cas9 to perform both CRISPRa/i and precise gene editing. While reprogramming of most DNA repair factors or their combinations tested enhanced HDR efficiency, simultaneously activatingCDK1and repressingKU80has the strongest effect with increased HDR rate upto an order of magnitude. Doxycycline-induced dgRNA-based CRISPRa/i programming of DNA repair enzymes, as well as viral packaging enabled flexible and tunable HDR enhancement for broader applicability in mammalian cells. Our study provides an effective, flexible, and potentially safer strategy to enhance precise genome modifications, which might broadly impact human gene editing and therapy.
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影响因子：
16.8
作者：
通讯作者：
--
影响因子：
64.8
作者：
Konermann S;Brigham MD;Trevino AE;Joung J;Abudayyeh OO;Barcena C;Hsu PD;Habib N;Gootenberg JS;Nishimasu H;Nureki O;Zhang F
通讯作者：
Zhang F
DOI：
10.1016/j.tig.2008.08.007
发表时间：
2008-11
期刊：
Trends in genetics : TIG
影响因子：
--
作者：
McVey M;Lee SE
通讯作者：
Lee SE
DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Mali P;Yang L;Esvelt KM;Aach J;Guell M;DiCarlo JE;Norville JE;Church GM
通讯作者：
Church GM
影响因子：
64.8
作者：
Dever DP;Bak RO;Reinisch A;Camarena J;Washington G;Nicolas CE;Pavel-Dinu M;Saxena N;Wilkens AB;Mantri S;Uchida N;Hendel A;Narla A;Majeti R;Weinberg KI;Porteus MH
通讯作者：
Porteus MH