CRISPR-Cas9 corrects sickle cell mutation
Collaborative effort to reduce off-target CRISPR-Cas9 genome editing
Several groups previously described Cas9 mutants with improved specificity; however, all show significantly reduced activity when used in the clinically relevant RNP format. These previous mutants were developed using intelligent design based on known protein crystal structures. Instead, IDT scientists used an unbiased method to screen approximately 250,000 random Cas9 mutants to identify those rare mutants that improved specificity without compromising activity. After several rounds of selection, a single mutant emerged, now known as Alt-R HiFi Cas9 nuclease, that provides the desired high on-target, low off-target characteristics. In the published study, a collaborative team from IDT and Stanford demonstrates the robust on-target editing and minimal off-target cleavage achieved by HiFi Cas9 in several therapeutically relevant loci in hard-to-edit HSPCs. They also show HiFi Cas9-mediated correction of the sickle cell disease-causing p.E6V mutation in patient-derived HSPCs.
Mark Behlke, MD, PhD, chief scientific officer at IDT and a co-author of the study, said, “Previous attempts at improving Cas9 specificity characterized 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. Prof. Porteus demonstrated utility using the new system to correct the SCD mutation in normal human blood-forming stem cells while minimizing known off-target activity. We anticipate significant interest in use of the new Cas9 mutant in translational medical applications.”
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