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

Abstract 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.