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CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis . This study greatly expands the targeting scope of Cas12a for crop genome engineering. CRISPR-Cas12a is a promising system for targeting AT-rich regions of the genome. Here the authors identify Cas12a orthologs with expanded targeting scope and develop a highly multiplexable editing system in rice.

Rice kernel smut (RKS), caused by the basidiomycete fungus Tilletia horrida , is a major disease impacting the production of male sterile lines of rice ( Oryza sativa ) at the globally. Despite its significance, the understanding of mechanisms underlying resistance to RKS remain limited. This study employed map-based cloning to isolate the CC-NBS-LRR (CNL) RKS resistance gene OsnTNB.11 from the resistant rice line Jiangcheng3B. The knockout of OsnTNB.11 in Jiangcheng3B resulted in plants significantly more susceptible to T. horrida than the wild type. Stable OsnTNB.11 overexpression transgenic rice plants developed resistance against T. horrida . We further found that the coiled-coil (CC) domain of OsnTNB.11 (OsnTNB.11-CC) interacted with and stabilised rice methionine synthetases OsMETS1, a key enzyme involved in ethylene (ET) biosynthesis. OsnTNB.11 activated the transcriptional levels of ET-related defence genes and enhance ET contents, whereas the exogenous application of ACC (an ethylene biosynthesis precursor) improved rice resistance to T. horrida . Further, the introduction of OsnTNB.11 into a disease-susceptible rice variety promoted resistance to RKS without impacting grain yield. In summary, we determined a regulatory model of a novel RKS resistance mediated via the ET signalling pathway. These results enhance our understanding of RKS resistance mechanisms and provide a valuable genetic resource for resistant T. horrida rice male sterile lines for breeding.

The application of genetically engineered (GE) crops in pest management raises biosafety concerns among governments, the scientific community, and the public, especially with the emergence of RNA interference (RNAi)-based crops expressing insecticidal double-stranded RNA (dsRNA). These crops may pose challenges to public health, agriculture, and conservation, and they could also present risks to non-target organisms, including beneficial natural enemies of pests. Natural enemies of insects are a significant component of global biodiversity and play a crucial role in managing insect pests within agroecosystems. This study addresses the biosafety concerns associated with insect-resistant transgenic dsRNA-expressing crops, focusing on their potential unintended effects on non-target organisms, particularly natural enemies. We combined biological and bioinformatic approaches, utilizing both food-chain delivery and animal-feeding systems, to comprehensively evaluate the potential unintended effects of exogenous insecticidal dsRNA expressed by dsAllim cotton on the biological parameters and transcriptome of the cotton-field predatory natural enemy, . The findings indicate that dsAllim cotton had no adverse effects on , suggesting its potential safety for non-target beneficial insects. At both developmental and transcriptomic levels, ds cotton showed no significant impact on . These results support the use of ds cotton as a reference in developing regulatory frameworks for the risk assessment of RNAi crops. Together with previous research, our findings underscore the importance of conducting RNAi crop safety evaluations for non-target organisms on a case-by-case basis, with particular attention to potential off-target effects.

Ustilago crameri is a pathogenic basidiomycete fungus that causes foxtail millet kernel smut (FMKS), a devastating grain disease in most foxtail-millet-growing regions of the world. Here, we report an assembled high-quality genome sequence of U. crameri strain SCZ-6 isolated from the diseased grains of foxtail millet in Changzhi, Shanxi Province, China. The genome size is 19.55 Mb, consisting of 73 contigs (N50 = 840,209 bp) with a G + C content of 54.09%, and encoding 6576 predicted genes and 6486 genes supported by RNA-seq. Evolutionarily, U. crameri lies close to the barley smut U. hordei, and an obvious co-linearity was observed between these two smut fungi. We annotated the genome of U. crameri strain SCZ-6 using databases, identifying 1827 pathogen–host interaction (PHI)-associated genes, 1324 genes encoding fungal virulence factors, 259 CAZy-related genes, 80 genes encoding transporters, and 206 putative cytochrome P450 genes; their expression profiles at different inoculation time points were also detected. Additionally, 70 candidate pathogen effectors were identified according to their expression patterns and predicted functions. In summary, our results provide important insights into the pathogenic mechanisms of the pathogenesis-related genes of U. crameri and a robust foundation for further investigation.

Rice kernel smut (RKS), caused by the fungus Tilletia horrida, has become a major disease in rice-growing areas worldwide, especially since the widespread cultivation of high-yielding hybrid rice varieties. The disease causes a significant yield loss during the production of rice male sterile lines by producing masses of dark powdery teliospores. This review mainly summarizes the pathogenic differentiation, disease cycle, and infection process of the T. horrida, as well as the decoding of the T. horrida genome, functional genomics, and effector identification. We highlight the identification and characterization of virulence-related pathways and effectors of T. horrida, which could foster a better understanding of the rice–T. horrida interaction and help to elucidate its pathogenicity molecular mechanisms. The multiple effective disease control methods for RKS are also discussed, included chemical fungicides, the mining of resistant rice germplasms/genes, and the monitoring and early warning signs of this disease in field settings.

Carbohydrate-binding modules (CBMs) are essential virulence factors in phytopathogens, particularly the extensively studied members from the CBM50 gene family, which are known as lysin motif (LysM) effectors and which play crucial roles in plant–pathogen interactions. However, the function of CBM50 in Tilletia horrida has yet to be fully studied. In this study, we identified seven CBM50 genes from the T. horrida genome through complete sequence analysis and functional annotation. Their phylogenetic relationships, conserved motifs, promoter elements, and expression profile were further analyzed. The phylogenetic analysis indicated that these seven ThCBM50 genes were divided into three groups, and close associations were observed among proteins with similar protein motifs. The promoter cis-acting elements analysis revealed that these ThCBM50 proteins may be involved in the regulation of the phytohormones, stress response, and meristem expression of the host plant during T. horrida infection. The transcriptome data indicated that four ThCBM50 genes were upregulated during T. horrida infection. We further found that ThCBM50_1 caused cell death in the leaves of Nicotiana benthamiana, and its signal peptide (SP) had a secreting function. These results offer important clues that highlight the features of T. horrida CBM50 family proteins and set the stage for further investigation into their roles in the interactions between T. horrida and rice.

Rice leaf folder, stem borer and brown planthopper (BPH) are the most devastating rice insect pests. Developing and planting insect-resistant rice varieties is the most economical and effective measure for controlling these pests. BPH can be controlled with native BPH-resistance genes in rice, while at present rice leaf folder and stem borer can only be controlled through planting transgenic Bt rice. In this study, the breeding of a new restorer line KR022 possessing stacked BPH-resistance genes Bph14 and Bph15, Bt gene cry1C and glufosinate-resistance gene bar, is reported for the first time. A rice restorer line R022 with BPH-resistance genes Bph14 and Bph15 was used as a recurrent parent to cross with the transgenic rice T1C-19 of cry1C and bar genes during the breeding process. The restorer line KR022 was developed from the backcross populations of R022 and T1C-19 through molecular marker-assisted selection and glufosinate-resistance selection. The cry1C and bar genes were found to integrate on chromosome 11 of KR022, and the genome recovery of KR022 was up to 95.8 % of the R022 genome. The quantification of Cry1C protein expression showed that it was expressed at different levels in the leaf, stem, panicle, endosperm, and root of KR022 and its hybrid rice. The insect-resistance evaluation indicated that KR022 and its hybrid rice had good resistance to rice leaf folder and stem borer, both in laboratory settings and in the field. Furthermore, they exhibited increased resistance to BPH at both the seedling and mature stage. The field trial showed there was no significant difference in key agronomic traits between KR022 and its recurrent parent R022, and four hybrids from KR022 yield much higher than the control II-You 838. Moreover, KR022 and its hybrid rice were found to have resistance to the herbicide glufosinate. These results demonstrate that KR022 is effective as a rice restorer line for the breeding of “green super rice”, possessing multiple tolerances to rice BPH, stem borer, leaf folder and glufosinate.

The rapid development of the CRISPR–Cas9, –Cas12a and –Cas12b genome editing systems has greatly fuelled basic and translational plant research 1 , 2 , 3 , 4 , 5 – 6 . DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G) 7 , limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant 8 . Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. 9 ) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for secondary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox. An engineered SpRY Cas9 variant enables efficient gene editing without PAM requirement in rice transgenic lines and Dahurian larch protoplasts, and its derived base editors can edit the rice genome efficiently in a PAM-less fashion too.

Summary Cytosine base editors (CBEs) are great additions to the expanding genome editing toolbox. To improve C‐to‐T base editing in plants, we first compared seven cytidine deaminases in the BE3‐like configuration in rice. We found A3A/Y130F‐CBE_V01 resulted in the highest C‐to‐T base editing efficiency in both rice and Arabidopsis. Furthermore, we demonstrated this A3A/Y130F cytidine deaminase could be used to improve iSpyMacCas9‐mediated C‐to‐T base editing at A‐rich PAMs. To showcase its applications, we first applied A3A/Y130F‐CBE_V01 for multiplexed editing to generate microRNA‐resistant mRNA transcripts as well as pre‐mature stop codons in multiple seed trait genes. In addition, we harnessed A3A/Y130F‐CBE_V01 for efficient artificial evolution of novel ALS and EPSPS alleles which conferred herbicide resistance in rice. To further improve C‐to‐T base editing, multiple CBE_V02, CBE_V03 and CBE_V04 systems were developed and tested in rice protoplasts. The CBE_V04 systems were found to have improved editing activity and purity with focal recruitment of more uracil DNA glycosylase inhibitors (UGIs) by the engineered single guide RNA 2.0 scaffold. Finally, we used whole‐genome sequencing (WGS) to compare six CBE_V01 systems and four CBE_V04 systems for genome‐wide off‐target effects in rice. Different levels of cytidine deaminase‐dependent and sgRNA‐independent off‐target effects were indeed revealed by WGS among edited lines by these CBE systems. We also investigated genome‐wide sgRNA‐dependent off‐target effects by different CBEs in rice. This comprehensive study compared 21 different CBE systems, and benchmarked PmCDA1‐CBE_V04 and A3A/Y130F‐CBE_V04 as next‐generation plant CBEs with high editing efficiency, purity, and specificity.