Modulation of Double Strand Breaks repair to promote Cas9 and PEn dependent DNA insertions 

The CRISPR-Cas9 system is a powerful tool for genome engineering, but the low efficiency of targeted gene integration is a significant challenge for therapeutic applications, especially in non-dividing cells.

We developed new approaches to enhance the efficiency of targeted integrations in mammalian cells. This includes 2HDR, a method using a mix of small molecule inhibitors to facilitate gene insertion in dividing cells, and PEn/2iPEn employing an RT/DNA polymerase-driven strategy to promote templated insertions via NHEJ-dependent and NHEJ-independent  pathway.

The use of the specified DNA repair inhibitors, combined with our newly developed nuclease, PsCas9, reduces the potential for unwanted on-target and off-target effects.