Recruitment of DNA Repair MRN Complex by Intrinsically Disordered Protein Domain Fused to Cas9 Improves Efficiency of CRISPR-Mediated Genome Editing

Abstract

CRISPR/Cas9 is a powerful tool for genome editing in cells and organisms. Nevertheless, introducing directed templated changes by homology-directed repair (HDR) requires the cellular DNA repair machinery, such as the MRN complex (Mre11/Rad50/Nbs1). To improve the process, we tailored chimeric constructs of Cas9, in which SpCas9 was fused at its N- or C-terminus to a 126aa intrinsically disordered domain from HSV-1 alkaline nuclease (UL12) that recruits the MRN complex. The chimeric Cas9 constructs were two times more efficient in homology-directed editing of endogenous loci in tissue culture cells. This effect was dependent upon the MRN-recruiting activity of the domain and required lower amounts of the chimeric Cas9 in comparison with unmodified Cas9. The new constructs improved the yield of edited cells when making endogenous point mutations or inserting small tags encoded by oligonucleotide donor DNA (ssODN), and also with larger insertions encoded by plasmid DNA donor templates. Improved editing was achieved with both transfected plasmid-encoded Cas9 constructs as well as recombinant Cas9 protein transfected as ribonucleoprotein complexes. Our strategy was highly efficient in restoring a genetic defect in a cell line, exemplifying the possible implementation of our strategy in gene therapy. These constructs provide a simple approach to improve directed editing.

RS‐1 enhances CRISPR‐mediated targeted knock‐in in bovine embryos

“…Further improvement of targeted KI efficiency may be achieved by other complementary approaches such as modified versions of the CRISPR system that employ Cas9 fused to the repair template (Aird, Lovendahl, St Martin, Harris, & Gordon, 2018; Savic et al, 2018) or to an MRN recruiter (Reuven, Adler, Broennimann, Myers, & Shaul, 2019), or by restricting DSB generation to those phases of the cell cycle when HDR is active: S/G2 (Hustedt & Durocher, 2016). Several approaches have been explored to restrict DSB formation to S/G2 phases such as the use of Cas9 fusion proteins with cell phase‐restricted activity (Charpentier et al, 2018; Gutschner et al, 2016) or cell cycle synchronizers (Zhang et al, 2017).…”

OsMre11 Is Required for Mitosis during Rice Growth and Development

“…If the MRX complex congregated in large numbers at the end of severely damaged chromosomes, it indicated that the complex is able to generate a response to the DNA damage [ 58 , 59 , 60 ]. In humans, targeted genome editing was performed in HEK293 cells, and the activation of DNA repair mechanisms, including MRN, could be induced [ 61 ]. In Arabidopsis , γ-irradiation caused the production of DSBs, which led to MRN-dependent activation of ATM and ATR kinases [ 12 ].…”

Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks

“…A novel strategy has been reported that uses chimeric Cas9 constructs, in which SpCas9 is fused to a 126-amino acid intrinsically disordered domain from the HSV-1 alkaline nuclease (UL12), which is not an HDR-relevant factor. In this approach, UL12 could increase the recruitment of the MRN complex to DSBs, thereby facilitating HDR [111].…”

“…Regarding 53BP1 antagonists, Pinder et al showed that cells overexpressing wild-type BRCA1 or its hyper-recombination mutant variants (BRCA1K1702M, BRCA1M1775R) also exhibited increased rates of Cas9-induced HDR [101]. In this approach, UL12 could increase the recruitment of the MRN complex to DSBs, thereby facilitating HDR [111]. Several HDR enhancers with yet unknown mechanisms of action on DSB repair have also been identified.…”