Efficient genome editing in rice with miniature Cas12f variants

Abstract

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 investigation of genome cleavage activity in rice, we unveiled a substantial enhancement in editing efficiency for both AsCas12f variants, particularly for AsCas12f-HKRA, which achieved an editing efficiency of up to 53%. Furthermore, our analysis revealed 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 significant 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.

Supplementary information: The online version contains supplementary material available at 10.1007/s42994-024-00168-2.

Keywords: AsCas12f; CRISPR/Cas; Cas12; Genome editing; Rice.

Fig. 1

CRISPR/AsCas12f-mediated genome editing in rice. A Schematic of AsCas12f and its two activity-enhanced variants, AsCas12f-YHAM and AsCas12f-HKRA, as well as the optimized sgRNA, sgRNA_ΔS3–5_v7. B Summary of editing efficiencies of the T0 transgenic rice plants. The target sequences in OsPDS, OsYSA, and OsD14 are shown, with PAM sequences highlighted in blue. C Albino leaves observed in OsPDS and OsYSA T0 plants. Scale bar, 0.5 cm. D Distribution of homozygous, biallelic, heterozygous, and chimeric mutations in OsPDS, OsYSA, and OsD14. E Frequencies of each deletion size in T0 plants. F Genotyping of OsPDS T0 edited plants using AsCas12f-HKRA variant. G, H Deletion pattern of AsCas12f, showing frequencies at each deletion position (G) and size (H). Deletion positions were defined as positions from which the deletions originated. Blue arrowheads represented reported cleavage sites (Wu et al. 2021). The frequencies for all deletion sizes are presented in Fig. S5. The heatmaps on the right indicate the percentage of corresponding left deletion size