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Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control. We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase (ALS) gene to confer herbicide tolerance in rice. The four different missense mutations in the P171 codon, P171S, P171A, P171Y and P171F, exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors. For example, P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides. Interestingly, a novel triple mutant (P171F/G628E/G629S) had the highest tolerance to all five tested herbicides. Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron. Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties. base editing, herbicide tolerance, rice, acetolactate synthase (ALS)

Mitogen-activated protein kinases (MAPKs) play essential roles in mediating biotic and abiotic stress responses in plants. However, the MAPK gene family in strawberry has not been systematically characterized. Here, we performed a genome-wide survey and identified 12 MAPK genes in the Fragaria vesca genome. Protein domain analysis indicated that all FvMAPKs have typical protein kinase domains. Sequence alignments and phylogenetic analysis classified the FvMAPK genes into four different groups. Conserved motif and exon-intron organization supported the evolutionary relationships inferred from the phylogenetic analysis. Analysis of the stress-related cis-regulatory element in the promoters and subcellular localization predictions of FvMAPKs were also performed. Gene transcript profile analysis showed that the majority of the FvMAPK genes were ubiquitously transcribed in strawberry leaves after Podosphaera aphanis inoculation and after treatment with cold, heat, drought, salt and the exogenous hormones abscisic acid, ethephon, methyl jasmonate, and salicylic acid. RT-qPCR showed that six selected FvMAPK genes comprehensively responded to various stimuli. Additionally, interaction networks revealed that the crucial signaling transduction controlled by FvMAPKs may be involved in the biotic and abiotic stress responses. Our results may provide useful information for future research on the function of the MAPK gene family and the genetic improvement of strawberry resistance to environmental stresses.

The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9 (SpCas9) and its variants. The detailed mechanism remains unknown. Here, based on in vitro cleavage assays and base editing analysis, we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1). We also show that these nicks are made on the target DNA strand. These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets. This system provides a novel tool for achieving trait stacking in plants.

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