Efficient genome editing in rice with miniature Cas12f variants

This study is led by Professor Junjie Tan (Nanjing Agricultural University, Nanjing, China). Genome editing, particularly using the CRISPR/Cas system, has revolutionized biological research and crop improvement. Despite the widespread use of CRISPR/Cas9, it faces limitations such as PAM sequence requirements and challenges in delivering its large protein into plant cells. The hypercompact Cas12f, derived from Acidibacillus sulfuroxidans (AsCas12f), stands out due to its small size of only 422 amino acids and its preference for a T-rich motif, presenting advantageous features over SpCas9. However, its editing efficiency is extremely low in plants. Recent studies have generated two AsCas12f variants, AsCas12f-YHAM and AsCas12f-HKRA, demonstrating higher editing efficiencies in mammalian cells, yet their performance in plants remains unexplored.

In this study, through a systematic exploration of genome cleavage activity in rice, the authors revealed a significant improvement in editing efficiency for both AsCas12f variants, with AsCas12f-HKRA showing particularly noteworthy enhancement, achieving an editing efficiency of up to 53.1%. The AsCas12f-HKRA variant exhibited superior compatibility with substrate sequences and demonstrated higher nuclease activity.

Moreover, upon analyzing each mutant allele, the authors unveiled that AsCas12f predominantly induces deletion in the target DNA, displaying a unique deletion pattern primarily concentrated at positions 12, 13, 23, and 24, resulting in deletion size mainly of 10- and 11- bp, suggesting substantial potential for targeted DNA deletion using AsCas12f. These findings expand the toolbox for efficient genome editing in plants, offering promising prospects for precise genetic modifications in agriculture.

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Efficient genome editing in rice with miniature Cas12f variant