Engineering an optimized hypercompact CRISPR/Cas12j-8 system for efficient genome editing in plants

Abstract

The Cas12j-8 nuclease, derived from the type V CRISPR system, is approximately half the size of Cas9 and recognizes a 5′-TTN-3′ protospacer adjacent motif sequence, thus potentially having broad application in genome editing for crop improvement. However, its editing efficiency remains low in plants. In this study, we rationally engineered both the crRNA and the Cas12j-8 nuclease. The engineered crRNA and Cas12j-8 markedly improved genome editing efficiency in plants. When combined, they exhibited robust editing activity in soybean and rice, enabling the editing of target sites that were previously uneditable. Notably, for certain target sequences, the editing activity was comparable to that of SpCas9 when targeting identical sequences, and it outperformed the Cas12j-2 variant, nCas12j-2, across all tested targets. Additionally, we developed cytosine base editors based on the engineered crRNA and Cas12j-8, demonstrating an average increase of 5.36- to 6.85-fold in base-editing efficiency (C to T) compared with the unengineered system in plants, with no insertions or deletions (indels) observed. Collectively, these findings indicate that the engineered hypercompact CRISPR/Cas12j-8 system serves as an efficient tool for genome editing mediated by both nuclease cleavage and base editing in plants.