The CRISPR-Cas9 system is a powerful technique for genome editing, in which Cas9, in complex with a single-guide RNA (sgRNA), creates a double-stranded break at a target site followed by the cell’s repair machinery making nucleotide changes in the DNA.

Though Streptococcus pyogenes Cas9 (SpCas9) has been successfully used in a variety of biological systems, genomic off-targeting remains a concern, particularly for clinical applications.

Researchers characterized a naturally occurring Francisella novicida Cas9 (FnCas9), and found that it demonstrates high specificity in recognizing and binding to its intended targets.

FnCas9’s specificity was mediated by reduced DNA binding affinity when mismatches to the target sgRNA are present, resulting in negligible binding to off-target loci differing by one or more mismatches.

The authors successfully used FnCas9 for in vivo genome editing, where the enzyme showed highly specific target recognition and negligible off-targeting, with higher homology-directed repair rates compared with SpCas9. The authors also showed that FnCas9 could be used to correct disease-causing mutations in patient-derived cells in a model of sickle cell anemia.

The authors suggest that the high specificity and minimal off-targeting of FnCas9 make it a suitable candidate for precise therapeutic genome editing, according to the authors.

https://www.pnas.org/content/early/2019/09/27/1818461116