Engineered Cas12j‐8 is a Versatile Platform for Multiplexed Genome Modulation in Mammalian Cells

Cas12j-8 is a compact Cas nuclease discovered from the metagenome of giant bacteriophages, consisting of only 717 amino acids and recognizing the '5-TTN-3' protospacer adjacent motif (PAM) sequence. However, its low gene editing efficiency in mammalian cells limits its application in therapeutic gene editing. To address this limitation, structure-guided mutagenesis is employed to replace key negatively charged residues with arginine, strengthening DNA binding. The resulting quintuple mutant, engineered Cas12j-8 (enCas12j-8), demonstrates robust on-target editing efficiency comparable to LbCas12a while maintaining low off-target effects. Cytosine base editors (CBEs) and adenine base editors (ABEs) are developed using enCas12j-8, achieving up to 29.54-fold C-to-T and 36.57-fold A-to-G conversion efficiency compared with the wild-type at the dominated sites, respectively. Notably, enCas12j-8 enables multiplexed editing of three genomic loci simultaneously via a single crRNA array, achieving efficiencies comparable to single-guide approaches. Additionally, enCas12j-8-ABE facilitates the disruption of splice acceptor sites, effectively inducing exon skipping in the SOD1 gene. This strategy holds potential significance for therapeutic genome modulation. These findings establish enCas12j-8 as a versatile, high-precision tool for genome engineering, combining efficient delivery, multiplexing capability, and compatibility with diverse editing modalities.