CRISPR–Cas9 is a widely used platform for plant genome editing, but its outcomes are typically dominated by small insertions and deletions (indels). Such limited mutation profiles restrict its utility in functional studies of non-coding RNAs and regulatory elements, such as microRNAs (miRNAs), untranslated regions (UTRs), and promoter sequences, where larger sequence disruptions are often required. Here, we developed enhanced exonuclease–Cas9 platforms, termed multiple nucleotide deletion Cas9 (MND–Cas9) systems, for efficient generation of large deletions in rice. By screening four exonucleases (RecJ, T5, TREX2, and SbcB), we established MND–Cas9v1 systems based on TREX2 or SbcB that produced substantially larger deletions without reducing editing efficiency. Further optimization with an inserted DNA-binding domain (DBD) between Cas9 and exonuclease yielded MND–Cas9v2, which simultaneously enhanced efficiency and deletion size. To expand PAM compatibility, we introduced PAM-relaxed Cas9–NG and SpG variants, generating MND–Cas9–NG/SpGv2 systems with broader targeting scope and superior performance compared to their parental nucleases. Finally, we demonstrated the utility of these systems in two applications: MND–Cas9v2 efficiently knocked out the miRNA gene OsMIR530, producing larger seeds, and generated extended deletions in the 3′UTR of OsGhd2, which upregulated its expression and increased grain size. These results demonstrate that MND–Cas9 systems enable high-efficiency generation of extended deletions and facilitate functional analyses of non-coding RNAs and regulatory sequences. Overall, this work establishes a versatile and expandable exonuclease–Cas9 platform that substantially broadens the mutational spectrum and application potential of CRISPR-Cas9 for plant genome engineering.

Casδ is a recently identified evolutionary transitional CRISPR system characterized by its compact size (~900 amino acids), broad temperature tolerance, and guidance with a short crRNA without the requirement of a tracrRNA. However, the low editing efficiency of Casδ in eukaryotic cells limits its application. Here, we have developed a hierarchical engineering strategy to improve the genome editing activity of Casδ‐1, with optimization focused on enhancing its interactions with the crRNA, the protospacer adjacent motif (PAM) duplex, the single‐ stranded DNA substrate, and the RNA–DNA heteroduplex. Through this strategy, we successfully generated an activity‐enhanced Casδ‐1 variant, designated enCasδ, which harbors 9 amino acid substitutions that synergistically augment its editing efficiency. In human cell lines, enCasδ showed 1.3‐ to 29.3‐fold higher editing activity than the wild‐type Casδ‐1 across ten tested genomic loci, with an average editing efficiency of 54.6%. In addition, enCasδ also mediated robust genome editing in maize; its editing efficiency increased by an average of 5.3‐fold relative to Casδ‐1, and reached up to an average of 80% at the TS4 and PSY1 loci in stable transgenic lines. The overall editing performance of enCasδ was comparable to that of Streptococcus pyogenes Cas9 (SpCas9) and other Cas12 nucleases. Collectively, enCasδ represents a highly optimized Casδ‐1 variant that broadens the applicability of the Casδ CRISPR system and facilitates robust genome editing in both animal cells and plants.

Cytosine base editors (CBEs) and adenine base editors (ABEs) are powerful tools for precise genome editing in plants. Conventionally, such base editors are built upon the CRISPR-Cas9 systems where Cas9 nickases are used. To expand the base editing scope and minimize off-target effects, base editors derived from the CRISPR-Cas12a systems are desired. However, the use of deactivated Cas12a (dCas12a) in such base editors constrains the editing activity, preventing the wide use of Cas12a base editors for plant research and trait development. In this study, we demonstrate the use of an ABE based on the efficient LbCas12a-RRV variant to introduce herbicide-resistant mutations in OsACCase in rice. To improve Cas12a CBEs and ABEs, we inserted introns into the coding sequence of dLbCas12a-RRV. This intron-containing Cas12a-CBE shows substantial improvement in editing efficiency in rice, compared to the non-intron counterparts. By contrast, the improvement of ABE with the intron-containing dLbCas12a-RRV is very limited, partly due to the already high baseline editing efficiency of the intron-less dLbCas12a-RRV ABE. Testing of these base editors in poplar shows elevated C-to-T base editing by dLbCas12a-RRV–intron-CBE. For A-to-G editing, ABEs built upon dLbCas12a-RV and dLbCas12a-RRV variants showed significant improvement over ABEs derived from wild-type LbCas12a and the ttLbCas12a variant. The addition of introns to dLbCas12a-RRV does not further improve the base editing efficiency. With whole genome sequencing in rice, we evaluated genome editing specificities with these improved Cas12a base editors. Our analyses show that both intron-containing Cas12a CBE and ABE barely introduce guide RNA-dependent off-target mutations. However, they can generate guide RNA-independent off-target mutations, which are likely attributed to the high enzymatic activities of the deaminases. Collectively, our study demonstrates the successful use of a Cas12a base editor for trait development and reports improved Cas12a CBEs and ABEs for precise base editing in plants.

Inter-organellar communication has emerged as a critical factor in maintaining cellular homeostasis under stress conditions. Chloroplasts function not only as central organelles for energy production but are also increasingly recognized as stress sensors and signal integrators. SIGMA FACTOR-BINDING PROTEIN 1 (SIB1), encoded by a nuclear gene, has been identified as a positive regulator of plant immunity, with localization in both chloroplasts and the nucleus. In this study, we identified Arabidopsis SEC23A, a COPII complex component known to mediate membrane trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus, as a novel interactor of SIB1. Our findings reveal that under normal conditions, both SIB1 and SEC23A localize to the ER, while SIB1 is also localized in the nucleus. Upon ER stress and/or treatment with the immunity inducer salicylate, both proteins are relocated from the ER to the chloroplasts. Notably, SEC23A, similar to SIB1, also functions as a positive regulator of disease resistance. In response to pathogen infection, SIB1 and SEC23A downregulate expression of chloroplast- and nucleus-encoded genes associated with photosynthesis while enhancing expression of defense-related genes. Together, our findings reveal a previously uncharacterized pathway of ER–chloroplast communication mediated by SIB1 and SEC23A during plant stress responses and immunity, providing novel insights into the intricate regulatory networks that govern inter-organellar communication under stress in plants.

Pre-messenger RNA (pre-mRNA) splicing is a critical mechanism for post-transcriptional regulation in plants. Through alternative splicing, plants produce diverse transcriptomes and proteomes that finely regulate development as well as responses to biotic and abiotic stresses. However, modulating the generation of specific splicing isoforms for functional characterization remains challenging, particularly in the non-model crop potato. Here, we show that two optimized TadA-derived base editors efficiently induce diverse mRNA splice variants by targeting specific splice sites. By evaluating multiple adenosine deaminases and performing multi-dimensional optimization, we developed an efficient adenine base editor RTF-ABE8e for potato. RTF-ABE8e achieved 100% editing efficiency at two StDL1 target sites in stable transgenic potato, with homozygous editing frequencies as high as 93.3% and 91.1%, respectively. We also developed RTF-TadDE, a dual-base editor based on a TadA-derived dual deaminase, for A-to-G and C-to-K (K = T/G) mutations in potato with an overall editing efficiency comparable to that of RTF-ABE8e. By targeting different splice sites with these base editors, we obtained diverse splicing isoforms carrying premature termination codons (PTCs) at StDL1 and StPDS and robust mutant phenotypes. These base editors enable efficient and precise editing of splice sites to trigger missplicing, making them powerful tools for manipulating splicing in plants.

Seed size is a critical factor in determining crop yield. Recent studies highlighted the importance of the MPK3/MPK6 cascade in regulating seed size in both Arabidopsis and rice. However, the underlying mechanism is not fully elucidated. Here, we report a novel function for the MKK4/MKK5–MPK3/MPK6 module in controlling the timing of endosperm cellularization, thereby regulating the seed size. Loss of function of MKK4/MKK5 or MPK3/MPK6 results in precocious endosperm cellularization and smaller seeds. WRKY transcription factor WRKY10/MINI3 is identified as a substrate of MPK3/MPK6 in this process. During early silique development, MINI3 is expressed and undergoes phosphorylation, a process mediated by MPK3/MPK6 at five specific serine residues. The phospho-deficient MINI3SA variant loses the function of wild-type MINI3 in vivo. Furthermore, we found that IKU2 and RLK7, two closely related receptor-like kinases, function upstream of the MKK4/MKK5–MPK3/MPK6–MINI3 pathway to regulate seed size. The phosphorylation of MINI3 mediated by MPK3/MPK6 enhances its transcriptional activity toward target genes, including IKU2, RLK7, and MINI3 itself, thereby forming a positive regulatory loop within this signaling pathway. In summary, our study elucidates a signaling pathway composed of IKU2/RLK7–MAPKKK(s)–MKK4/MKK5–MPK3/MPK6–MINI3 that regulates endosperm cellularization and seed size, providing a theoretical basis for crop breeding.