Characterization and engineering of highly efficient Cas12j genome editors

The large size of widely used CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) enzymes limits their delivery for therapeutic applications. Cas12j nucleases offer a hypercompact alternative but show modest editing efficiency. To overcome this limitation, we identified eight novel Cas12j orthologs from viral metagenomes, which in their native form exhibit low editing activity in mammalian cells. We therefore engineered T5 exonuclease-Cas12j fusions, resulting in substantially enhanced genome-editing activity across multiple mammalian cell types, reaching levels comparable to established compact CRISPR-Cas editors. Intriguingly, robust cellular editing occurred in the presence of a previously unrecognized trinucleotide sequence context within the target DNA. Furthermore, we developed Cas12j-based adenine base editors by coupling catalytically inactive Cas12j orthologs with adenine deaminase, enabling efficient A-to-G base conversion in mammalian cells. This study expands the CRISPR toolbox by establishing engineering principles that convert compact Cas12j nucleases into efficient and modular genome-editing platforms well suited for delivery-constrained therapeutic applications.