Systematic High Throughput Evaluation Reveals FrCas9 Superior Specificity and Efficiency for Therapeutic Genome Editing

CRISPR-Cas9 systems have shown great promise for genome editing, but their clinical applications are limited by off-target effects. This study systematically evaluates FrCas9, a CRISPR variant from Francisella novicida, comparing it with SpCas9 and OpenCRISPR-1 for therapeutic genome editing applications. Using high-throughput sequencing approaches including AID-seq, Amplicon sequencing, and GUIDE-seq, we assessed on-target efficiency and off-target profiles across multiple genomic loci. Our analysis examined over 1,800 target sites in human and mouse cells, with a particular focus on 21 genes relevant to cell and gene therapy (CGT). The study also investigated strategies to enhance FrCas9's performance through sgRNA optimization and TREX2 fusion. We found that FrCas9, especially when fused with TREX2, demonstrated superior specificity and reduced translocation rates compared to other systems, making it a promising tool for precise therapeutic genome editing. The sequencing data presented here provides comprehensive insights into the targeting specificity and efficiency of these CRISPR systems.