Increasing the efficiency of Cas9

Nucleic acid specialists Integrated DNA Technologies (IDT) have collaborated with Stanford University to describe a novel Cas9 mutant that shows improved specificity and maintains high activity.

In a study published in Nature Medicine, researchers at IDT and the laboratory of Professor Matthew Porteus at Stanford University show that in the medically relevant ribonucleoprotein (RNP) format the nutant shows improved specificity. Potential for medical use of the new mutant enzyme was demonstrated in human hematopoietic stem and progenitor cells (HSPCs), where it was able to correct the mutation in the beta-hemoglobin gene responsible for sickle cell disease.

Mark Behlke, MD, PhD, chief scientific officer at IDT and a co-author of the study, said, “Previous attempts at improving Cas9 specificity characterised the mutants using plasmid-based methods that result in sustained overexpression of the Cas9 protein, which increases off-target activity and is not ideal for medical applications.

“This sustained overexpression, however, rescued function of the mutants that otherwise showed low activity when used in the more transient RNP format. We specifically performed a broad screen to identify a mutant that performs well when used at the lower protein levels achieved with RNP delivery, maximizing safety and further reducing unwanted side effects.”

The novel HiFi Cas9 nuclease is n commercially available as Alt-R HiFi Cas9 Nuclease V3.