Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 119, Issue 49, Pages -Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2204259119
Keywords
CRISPR; genome editing; chromatin; epigenetics
Categories
Funding
- Centers for Excellence in Genomic Science of the National Institutes of Health [RM1HG009490]
- National Institute of General Medical Sciences [F32GM142146-01]
- Jane Coffin Childs Memorial Fund for Medical Research
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The fusion of PRDM9 and Cas9 in CRISPR-Cas genome editing can increase the efficiency of homology-directed repair (HDR) and avoid off-target editing.
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 method 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 H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency.
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