Altered DNA Repair Pathway Engagement by Engineered CRISPR-Cas9 Nucleases

Technology 

The engineered Cas nuclease (vCas9) is introduced into cells, where it decreases, inhibits, or prevents indel-producing DNA repair pathways, such as NHEJ, and enhances precise editing pathways like HDR and MMEJ. This shift is driven by specific mutations within the DNA binding cleft of the Cas nuclease. The technology is versatile, functioning in both dividing and non-dividing cells, making it suitable for a wide range of genome editing applications. 

Problem Addressed 

This technology addresses the limitations of traditional CRISPR-Cas systems, which frequently result in unpredictable and imprecise genome edits due to the common occurrence of indel mutations through pathways like NHEJ. These indels can lead to unintended genetic modifications, complicating the editing process. By decreasing, inhibiting, or preventing NHEJ and promoting HDR and MMEJ, this technology enables more precise and reliable genome edits. 

Advantages 

• The engineered Cas nucleases reduce NHEJ while increasing HDR and MMEJ, leading to more precise genome editing. 
• These nucleases are effective in both dividing and non-dividing cells, broadening their applicability. 
• By minimizing indel formation, the engineered nucleases reduce the likelihood of unintended mutations, enhancing the reliability of genome editing. 
• The mutations in the DNA binding cleft of the Cas nucleases improve the efficiency of precise genome edits.