Ye Lupeng, Wang Chengkun, Hong Lingjuan, Sun Ninghe, Chen Danyang, Chen Sidi, Han Feng
1Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, 310058 Hangzhou, China.
System Biology Institute, Integrated Science & Technology Center, 850 West Campus Drive, Room 361, West Haven, CT 06516 USA.
Cell Discov. 2018 Jul 24;4:46. doi: 10.1038/s41421-018-0049-7. eCollection 2018.
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 activating and repressing has 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.
CRISPR系统已被证明是用于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引导的核酸酶会引入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，而HDR是精确基因靶向所必需的另一种途径。已经开发了多种方法来提高HDR，包括化合物或RNA干扰介导的对NHEJ因子的抑制、HDR酶的小分子激活或CRISPR复合物的细胞周期定时递送。然而，这些方法面临多重挑战，效果中等或各不相同。在此，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。对NHEJ和HDR途径成分（如CtIP、CDK1、KU70、KU80和LIG4）的操纵由催化失活的引导RNA（dgRNA）介导，因此仅依靠单个具有催化活性的Cas9来执行CRISPRa/i和精确基因编辑。虽然对大多数测试的DNA修复因子或其组合进行重新编程可提高HDR效率，但同时激活和抑制具有最强的效果，HDR率提高了一个数量级。强力霉素诱导的基于dgRNA的DNA修复酶的CRISPRa/i编程以及病毒包装能够灵活且可调节地增强HDR，从而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，这可能会对人类基因编辑和治疗产生广泛影响。
