Abstract
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 CDK1 and repressing KU80 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.
Highlights
Organisms evolved multiple mechanisms to maintain genome integrity[1,2]
To enhance homologydirected repair (HDR) efficiency of clustered regularly interspaced palindromic repeats (CRISPR)-mediated gene editing with clean genetic approaches that avoid the potential side effects from chemical compounds, we leverage a method that tunes the expression of DNA damage repair pathway components by dead guide RNAs (dgRNAs)/active
The results showed robust activation and repression of both exogenous reporter genes and endogenous genes, where the EGFP’s mRNA level was significantly upregulated by dgGFP - MS2:MCP - P65-HSF1 (MPH) and repressed by dgGFP-Com:CK (Supplementary Fig. S1a–c), and the transcriptional level of ASCL1 and HBG1 were dramatically upregulated by dgRNA-MS2:MPH systems with gene-specific dgRNAs (Supplementary Fig. S1d, e)
Summary
As the cellular genome is constantly exposed to environmental damage, multiple DNA damage repair pathways exist to protect the genome from harmful or potentially catastrophic alterations[3]. DNA doublestrand break (DSB) repair pathways are highly conserved. Non-homologous DNA end joining (NHEJ) and homology-directed repair (HDR) are two major DNA repair pathways that can either act in concert or antagonistic manner[6]. HDR is a pathway which uses template. DNA, such as an intact sister chromosomal copy or an exogenous donor to repair the DSBs7, can robustly generate perfect repair[8]. NHEJ has been considered the major pathway to repair the DNA13, whereas HDR is more common in the Saccharomyces cerevisiae[14].
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.