This technology is based on a genetically engineered variant of the DNA repair factor RAD18 (“e18”) that has the ability to stimulate the precise repair of the Cas9-induced double-strand DNA breaks, thereby increasing the efficiency of CRISPR-mediated gene editing.
CRISPR/Cas9 technology has transformed gene editing for life science research and clinical intervention. A critical step in CRISPR-mediated gene editing is the repair of Cas9-induced double-strand DNA breaks via homology-directed repair (HDR), an endogenous cellular DNA repair process. However, the efficiency of this process is limited by competing mechanisms, such as non-homologous end joining. There is a need to improve the efficiency of HDR, in order to enhance efficacy of CRISPR-mediated gene editing.
This technology identifies RAD18, an E3 ubiquitin ligase that increases the frequency of HDR when overexpressed. The authors further engineer RAD18 to enhance the efficiency and specificity of the HDR function by deleting a functional domain of RAD18. They rename this engineered factor with enhanced HDR function as e18 (enhanced RAD18). The authors dissect the mechanism of action of e18 by demonstrating its ability to block the recruitment of 53BP1, a key inhibitor of HDR, to double-strand breaks. They demonstrate that e18 stimulates HDR by inhibiting NHEJ. In experiments using multiple human cell lines and both single-stranded as well as double-stranded DNA donor templates, e18 overexpression led to a two-fold increase in successful HDR events at several endogenous loci. The expression of e18 did not cause any toxicity in successfully edited human embryonic stem cells. This technology has potential to greatly enhance the efficiency of CRISPR-mediated gene editing.
Patent Pending
IR CU17329
Licensing Contact: Cynthia Lang