Abstract Common unintended chromosomal alterations induced by CRISPR/Cas9 in mammalian cells, particularly on-target large deletions and chromosomal translocations present a safety challenge for genome editing. Base editing and prime editing that can precisely introduce desired edits without double-stranded breaks and exogenous DNA templates face their own challenges. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified T4 DNA polymerase derived from phage T4 that greatly prevents undesired on-target large deletions and chromosomal translocations while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency to correct common frameshift mutations in DMD (exon 52 deletion) and restored higher level of dystrophin expression than Cas9-alone in human induced pluripotent stem cell-derived cardiomyocytes. Moreover, CasPlus can greatly reduce the frequency of on-target large deletions in mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus represses chromosomal translocations while maintaining gene disruption efficiency that is higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications.

Phage DNA polymerase prevents deleterious on-target DNA damage and enhances precise CRISPR/Cas9 editing