Journal
BIOMOLECULES
Volume 9, Issue 10, Pages -Publisher
MDPI
DOI: 10.3390/biom9100584
Keywords
Gene targeting; CRISPR/Cas9; homology-directed repair; genome editing; endogenous mutagenesis in cell lines
Categories
Funding
- Israel Science Foundation [1591/15]
- BLG Trust
- Emil and Rita Weissfeld Family Foundation
- Audrey and Sheldon Durst
- David M. Polen Charitable Trust
- Rabina Foundation
- Jean-Jacques Brunschwig Fund for the Molecular Genetics of Cancer
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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.
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