Abstract
CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile way to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K4me3 and H3K36me3 shown to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in DNA to double the observed HDR efficiency and increase the HDR:indel ratio by 3-fold compared to that induced by Cas9 alone. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin structure for the choice of DNA repair pathway during CRISPR-Cas genome editing and provide a new strategy to increase the efficiency of HDR.
Significance Statement CRISPR-Cas-mediated homology-directed repair (HDR) enables precision genome editing for diverse research and clinical applications, but HDR efficiency is often low due to competing end-joining pathways. Here, we describe a simple strategy to influence DNA repair pathway choice and improve HDR efficiency by engineering CRISPR-Cas9-methyltransferase fusion proteins. This strategy highlights the impact of histone modifications on DNA repair following CRISPR-Cas-induced double-stranded breaks and adds to the CRISPR genome editing toolbox.
Competing Interest Statement
The Regents of the University of California have patents issued and pending for CRISPR technologies on which J.A.D. is an inventor. J.A.D. is a cofounder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, Intellia Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Vertex, Caribou Biosciences, Intellia Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Algen Biotechnologies, Felix Biosciences, The Column Group, and Inari. J.A.D. is Chief Science Advisor to Sixth Street, a Director at Johnson & Johnson, Altos and Tempus, and has research projects sponsored by Apple Tree Partners and Roche.