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Disruption of susceptibility (S) genes in crops is an attractive breeding strategy for conferring disease resistance

Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transition. However, the molecular mechanism underlying the gradual activation of TaVRN1 during the vernalization process remains unknown. In this study, we identified the novel transcript VAS (TaVRN1 alternative splicing) as a non-coding RNA derived from the sense strand of the TaVRN1 gene only in winter wheat, which regulates TaVRN1 transcription for flowering. VAS was induced during the early period of vernalization, and its overexpression promoted TaVRN1 expression to accelerate flowering in winter wheat. VAS physically associates with TaRF2b and facilitates docking of the TaRF2b-TaRF2a complex at the TaVRN1 promoter during the middle period of vernalization. TaRF2b recognizes the Sp1 motif within the TaVRN1 proximal promoter region, which is gradually exposed along with the disruption of a loop structure at the TaVRN1 locus during vernalization, to activate the transcription of TaVRN1. The tarf2b mutants exhibited delayed flowering, whereas transgenic wheat lines overexpressing TaRF2b showed earlier flowering. Taken together, our data reveal a distinct regulatory mechanism by which a long non-coding RNA facilitates the transcription factor targeting to regulate wheat flowering, providing novel insights into the vernalization process and a potential target for wheat genetic improvement.

MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) family with a low level of homology to canonical Cas12a nucleases. It has been publicly released as a royalty-free nuclease for both academic and commercial use. Here, we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences (YTTN) in plants. Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-LbCas12a system. We develop two variants, MAD7-RR and MAD7-RVR that increase the target range of MAD7, as well as an M-AFID (a MAD7-APOBEC fusion-induced deletion) system that creates predictable deletions from 5′-deaminated Cs to the MAD7-cleavage site. Moreover, we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs. Using the CRISPR-MAD7 system, we have obtained regenerated mutant rice and wheat plants with up to 65.6% efficiency.

We describe here a CRISPR simultaneous and wide-editing induced by a single system (SWISS), in which RNA aptamers engineered in crRNA scaffold recruit their cognate binding proteins fused with cytidine deaminase and adenosine deaminase to Cas9 nickase target sites to generate multiplexed base editing. By using paired sgRNAs, SWISS can produce insertions/deletions in addition to base editing. Rice mutants are generated using the SWISS system with efficiencies of cytosine conversion of 25.5%, adenine conversion of 16.4%, indels of 52.7%, and simultaneous triple mutations of 7.3%. The SWISS system provides a powerful tool for multi-functional genome editing in plants.

Additional file 1: Figure S1. Flow cytometry of BFP-to-GFP conversion induced by PBE and the five PBEcs in rice protoplasts. Figure S2. Engineering the secondary structures of CRISPR RNA scaffolds. Figure S3. Flow cytometry of BFP-to-GFP conversion induced by various scRNAs and their cognate PBEcs in rice protoplasts. Figure S4. Frequencies of base editing of endogenous genes by different scRNAs and cognate PBEcs in rice protoplasts. Figure S5. Activities of esgRNA-2×MS2, esgRNA-3×MS2, sgRNA4.0, and esgRNA-2×com with cognate PBEcs in rice protoplasts. Figure S6. C-to-T editing frequencies generated by scaffold RNA-recruited APOBEC1 narrow-window variants in rice protoplasts. Figure S7. Flow cytometry of mGFP-to-GFP conversion induced by PABE and the three PABEcs in rice protoplasts. Figure S8. Flow cytometry of mGFP-to-GFP conversion induced by various scRNAs and their cognate PABEcs in rice protoplasts. Figure S9. Activities of the selected scaffold RNAs with their cognate PABEcs in rice protoplasts. Figure S10. Schematic of multiple sgRNAs assembly for SWISSv1.1 and SWISSv1.2. Figure S11. The distributions of deletion reads among the indel sequencing reads for SWISSv1.1, SWISSv1.2, and SWISSv3. Figure S12. Schematic of multiple sgRNAs assembly for SWISSv2 and SWISSv3. Figure S13. Comparison of the editing efficiencies between the SWISS systems and the individual genome editing tools (PBE, PBEc4, PABE-2, PABEc5, and paired nCas9). Figure S14. Simultaneous CBE, ABE, and DSB formation in rice plants. Table S1. The sgRNA sequences used to compare the activities of PBEcs and PABEcs. Table S2. The sgRNA sequences used for SWISSv1.1, SWISSv1.2, SWISSv2, and SWISSv3 editing in rice protoplasts. Table S3. Potential off-target sites analyzed for OsALS-T2, OsACC-T2, OsBADH2-Indels-sgL, and OsBADH2-Indels-sgR triple mutants. Table S4. Statistics of whole genome sequencing analysis. Table S5. Primer sequences used in this study.

Additional file 3. Review history.

Additional file 2: Sequences S1. DNA sequences of the OsU3 and TaU6 promoters used in this study. Sequences S2. DNA sequences of sgRNA, esgRNA, and scRNAs used in this study. Sequences S3. DNA sequences of the modules composed of PBEcs and PABEcs used in this study. Sequences S4. DNA sequences of templates for assemble multiple sgRNAs used in this study. Sequences S5. DNA sequences of SWISSv2/v3 and SWISSv2/v3-NG used in this study.