Expansion of the prime editing modality with Cas9 from Francisella novicida

Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase (RT) after nick formation by CRISPR nickase. In this study, we developed a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas 9 [FnCas9(H969A)] nickase module, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing was dramatically extended, and accurate prime editing was induced specifically for the target genes.