Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption

Because of their smallness, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing. Overall design: We employed primer-extension-mediated sequencing (PEM-seq) to study the gene-editing outcomes for various CRISPR-Cas nuclease, include: SpCas9, AsCas12a, LbCas12a, Un1Cas12f1_ge4.1, CasMINI,CasMINI_ge4.1, AsCas12f1, PlmCas12e, and PlmCas12e-R1-v2. Twelve different editing site were targeted for each nuclease.