Enhanced Cas12i3 system enables precise OsAUX3 editing for rice grain improvement

Abstract

An optimized Cas12i3 genome‐editing system enables highly efficient and predictable editing of regulatory sequences in rice. Precise promoter engineering fine‐tunes gene expression, improves grain size, and enhances production potential, demonstrating a powerful new approach for crop improvement through targeted regulation rather than gene disruption.

The rapid development of CRISPR‐Cas technologies has transformed plant functional genomics and precision breeding. Among diverse CRISPR nucleases, the compact type V–I nuclease Cas12i3 (~1,100 aa) is attractive for crop engineering due to its small size, which facilitates delivery via size‐limited vectors and multi‐effector T‐DNA constructs (Lai et al., 2021). In addition, Cas12i3 generates staggered DNA double‐strand breaks that preferentially produce multi‐nucleotide deletions, resembling Cas12a (Tang et al., 2017), but distinct from the blunt‐end cleavage of Cas9 (Zhang et al., 2026). However, its activity in rice (Oryza sativa) is limited (Lai et al., 2021). Although protein engineering has produced the high‐activity Cas12i3‐5M variant (S7R, D233R, D267R, N369R, and S433R) (Figures 1A, S1A; Duan et al., 2024), whether crRNA expression constrains its performance remains unclear. Here, we identify crRNA abundance as a key bottleneck and develop an enhanced Cas12i3‐5M‐CP system for OsAUX3 promoter editing and rice improvement.

Figure 1.

Optimization of Cas12i3 systems and application to OsAUX3 promoter engineering for rice improvement

(A) AlphaFold3‐predicted structures of wild‐type Cas12i3 and Cas12i3‐5M. (B) T‐DNA architectures of Cas12i3, Cas12i3‐5M, and Cas12i3‐5M‐CP. (C) Editing efficiencies of the three systems at 24 endogenous rice loci in protoplasts. (D) Violin plots showing editing‐efficiency distributions and medians from (C). (E) Relative accumulation of Cas12i3‐5M transcripts and mature crRNAs in protoplasts determined by RT‐qPCR. (F) Effects of PAM preferences (VTTV vs. VTTTV) on editing by Cas12i3‐5M and Cas12i3‐5M‐CP. (G, H) Distributions of deletion positions relative to the PAM (G) and deletion sizes (H) across the 24 target sites. (I) Mutation profiles of T0 rice lines generated by Cas12i3‐5M‐CP at the OsAUX3 promoter; the OsARF4 transcription factor‐binding site (TFBS) is highlighted in red. (J) Representative grain morphology of OsAUX3 promoter‐edited and wild‐type (WT) plants (scale bar = 1 cm). (K) Relative expression of OsAUX3 in WT and mutant panicles. (L) Grain length of WT and OsAUX3 mutants. (M) Plant architecture of WT and OsAUX3 mutants at the reproductive stage (scale bar = 30 cm). (N) Schematic model showing that deletion of the repressive motif in the OsGLW7 5′UTR increases OsGLW7 transcript abundance. (O) Editing efficiencies of different Cas12i3 systems at the OsGLW7 locus in rice protoplasts. (P) Relative OsGLW7 expression in untreated protoplasts and protoplasts edited by the three Cas12i3 systems. Each dot represents an independent biological replicate. Data are means ± SD. Statistical significance was assessed by two‐tailed unpaired t‐tests: ns, P > 0.05; **P < 0.01. In violin plots, solid and dashed lines indicate the median and quartiles, respectively.

To optimize Cas12i3‐mediated editing, we compared three expression architectures: wild‐type (WT) Cas12i3, Cas12i3‐5M, and Cas12i3‐5M‐CP (Figure 1B). In Cas12i3‐5M‐CP, crRNA is driven by a 35S‐CmYLCV‐U6 composite promoter (CP), previously shown to enhance guide RNA expression in rice (Jiang et al., 2020; Wang et al., 2025). We evaluated 24 endogenous rice loci containing VTTTV or VTTV PAMs (Figure S1B). WT Cas12i3 showed 2.64%–36.91% editing efficiency, Cas12i3‐5M achieved 9.26%–73.13% editing efficiency, and Cas12i3‐5M‐CP achieved up to 89.57% editing efficiency (Figure 1C, D), indicating that both nuclease activity and crRNA expression determine editing efficiency. Further, RT‐qPCR showed similar Cas12i3‐5M transcript levels between systems, whereas mature crRNA abundance increased approximately 1.8‐fold in Cas12i3‐5M‐CP (Figure 1E), identifying crRNA availability as a key limiting factor.

A broad targeting scope is essential for genome editing applications. Previous studies suggested that Cas12i3 activity in plants is largely restricted to VTTTV PAMs (Wang et al., 2025). However, analysis of 24 loci showed that Cas12i3‐5M‐CP, while slightly preferring VTTTV PAMs, also efficiently edits VTTV sites including ATTA, CTTA, and GTTA (Figures 1F, S2), expanding the targetable space in the rice genome, particularly in T‐rich regulatory regions. Deep sequencing revealed deletions mainly 6–15 bp (Figures 1H, S3–S5), centered 9–11 bp downstream of the PAM (Figure 1G), indicating a preference for medium‐sized deletions suitable for regulatory engineering. Off‐target analysis using Cas‐OFFinder (Bae et al., 2014) and deep sequencing in rice protoplasts (Figure S6) detected no mutations in WT Cas12i3, Cas12i3‐5M, or Cas12i3‐5M‐CP.

To demonstrate the practical utility, we targeted the OsAUX3 promoter, which contains an OsARF4 transcription factor‐binding site (TFBS) required for proper expression during grain filling (Qiao et al., 2021; Figure 1I). Following Agrobacterium‐mediated transformation, we generated multiple biallelic T0 lines. Sanger sequencing confirmed that representative mutants, including OsAUX3‐m01 and ‐m02, carried 6–12 bp deletions that disrupted the OsARF4‐binding site. These mutations significantly reduced OsAUX3 expression (Figure 1K) and increased grain length (Figure 1J, L). In contrast, panicle number, plant height, grain width, and overall plant architecture were unaffected (Figure 1M), indicating that precise disruption of a single cis‐regulatory element can improve grain size without detectable pleiotropic effects commonly associated with gene knockout strategies. Consistent with the T0 generation, T1 lines retained increased grain length and reduced OsAUX3 expression with unchanged grain width (Figure S7), demonstrating stable inheritance.

To evaluate whether the optimized Cas12i3 can also activate gene expression through noncoding sequence engineering, we targeted the grain‐size regulator OsGLW7. Its 5′UTR contains a tandem 2 × ‘CACTTC’ cis‐regulatory motif whose deletion increases OsGLW7 expression (Figure 1N; Si et al., 2016). A crRNA spanning this motif was designed, and editing activities of WT Cas12i3, Cas12i3‐5M, and Cas12i3‐5M‐CP were compared in rice protoplasts. Cas12i3‐5M and Cas12i3‐5M‐CP showed higher editing efficiency than WT Cas12i3 (Figure 1O), and 5′UTR motif edited plants showed increased OsGLW7 expression (Figure 1P), demonstrating that Cas12i3 enables both transcriptional repression and activation through noncoding sequence engineering.

The successful application of the Cas12i3‐5M‐CP platform has two important implications for crop precision breeding: (i) crRNA abundance is a general bottleneck for compact CRISPR systems (Liu et al., 2022; Zhang et al., 2024) and (ii) the predictable deletion profile enables regulatory element engineering for fine‐tuning gene expression in complex traits.

In conclusion, we developed Cas12i3‐5M‐CP with high efficiency, expanded PAM compatibility, and predictable mutational outcomes in rice. Its application in OsAUX3 promoter engineering and grain size improvement demonstrates its potential for precision molecular breeding. This compact system expands the CRISPR toolbox for crop improvement.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

X.T. and Y.Z. conceived the project and designed the experiments. R.Z. and X.T. generated the constructs and analyzed protoplast editing. R.Z. and X.Y. performed rice transformation and analyzed editing in stable lines. R.Z., X.T., and Y.Z. conducted data analysis, prepared the figures, and wrote the manuscript with input from all authors. All authors have reviewed and approved the final manuscript.

Supporting information

Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.70337/suppinfo

Supporting File 1