Artificial evolution of OsEPSPS through an improved dual cytosine and adenine base editor generated a novel allele conferring rice glyphosate tolerance

Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here,through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore,we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn(D213N)mutation(OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.

水稻冷胁迫诱导的甲基化差异片段CIDM7的分离和分析

采用基于AFLP的甲基化敏感扩增多态性的方法对5℃冷处理48h的水稻9311叶片DNA胞嘧啶甲基化模式进行了初步研究,发现冷处理48h后9311基因组中一些CCGG位点发生了重新甲基化或去甲基化,并获得一些与水稻cDNA高度同源的甲基化差异片段,其中CIDM7(coldinducingdifferentialmethylation)片段在冷胁迫后去甲基化。Northern杂交证明CIDM7在冷胁迫后增强表达。通过在日本晴全长cDNA数据库中的同源搜索获得其全长cDNA序列,该cDNA序列全长1422bp,编码425个氨基酸,经氨基酸序列分析为一个具有Fbox结构的假定蛋白。CIDM7为单拷贝,定位于日本晴10号染色体12.56～12.57Mb。

Diverse nucleotide substitutions in rice base editing mediated by novel TadA variants

CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes.Compared with the welldeveloped adenine base editors(ABEs),cytosine base editors(CBEs)and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice,significantly limiting their application.Here,we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice.We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID*D,and TadA-CDd outperformed TadA-E27R/N46L.A C-to-G base editor(CGBE)engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P.In addition,a dual base editor constructed with a single protein,TadDE,enabled simultaneous,highly efficient C-to-T and A-to-G editing in rice.Collectively,our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice,providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.

Differentially methylated genomic fragments related with sexual dimorphism of rice pests, Sogatella furcifera

Sogatella furcifera （Hovarth） is a major rice pest with sexual dimorphism. The objective of the current research was to monitor differentially cytosine methylation at CCGG sequences in male and female adults of S. furcifera to determine the association between gene methylation and sexual phenotypes using methylation-sensitive representa- tional difference analysis. After the second subtractive hybridization, four differentially methylated DNA bands were obtained and sequenced. Ten different fragments were found. One fragment from the positive hybridization was 120 bp, and highly similar to the tram- track genes from Nasonia vitripennis. Another fragment from the reverse hybridization was 414 bp, and homologous to the 28S rRNA gene of S. furcifera with a similarity rate as high as 99%. We also discussed how DNA methylation of tramtrack and 28S rRNA genes produced effects on sexual differentiation and development. These results provide potential evidence that DNA methylation of some genes may be related to sexual phenotype variations in S.furcifera and will facilitate future studies on the epigenetic mechanisms of insect sexual dimorphism.

Novel Wx alleles generated by base editing for improvement of rice grain quality

Amylose content(AC),which is regulated by the Waxy(Wx)gene,is a major indicator of eating and cooking quality(ECQ)in rice(Oryza sativa).Thus far,only a limited number of mutations in the N-terminal domain of Wx were found to have a major impact on the AC of rice grains and no mutations with such effects were reported for other regions of the Wx protein.Here,nucleotide substitutions in the middle region of Wx were generated by adenine and cytosine base editors.The nucleotide substitutions led to changes in 15 amino acid residues of Wx,and a series of novel Wx alleles with ACs of 0.3%-29.43%(wild type with AC of 19.87%)were obtained.Importantly,the waxy~(abe2)allele showed a"soft rice"AC,improved ECQ,favorable appearance,and no undesirable agronomic traits.The transgenes were removed from the waxy~(abe2)progeny,generating a promising breeding material for improving rice grain quality.

水稻Osrdr1突变体全基因组CCGG位点甲基化的研究

DNA胞嘧啶甲基化变异调控植物基因组内功能基因表达和转座子转座活性,影响植物生长和发育的稳定性。RNA-dependent RNA polymerase 2(RDR2)基因参与RNA-directed DNA methylation(RdDM)途径指导合成CHH(H=G、T、A)甲基化,CHH甲基化在基因和转座子上表现不同,是目前DNA胞嘧啶甲基化的研究热点之一。拟南芥中RDR1基因与RDR2属于同一家族,最新研究结果证实,RDR1基因不仅可以指导CHH甲基化形成,并且其功能缺失会不同程度影响CG和CHG甲基化。前期研究工作表明,水稻中其同源基因OsRDR1功能缺失可在特异位点影响DNA胞嘧啶甲基化,但是否大规模影响全基因组DNA甲基化尚不明确。在此基础上,采用甲基化敏感多态性扩增(Methylation sensitive amplification polymorphism,MSAP)技术研究OsRDR1基因功能缺失突变体全基因组CCGG位点的甲基化情况。结果同野生型比较分析发现,OsRDR1基因功能缺失没有引起全基因组CCGG位点甲基化水平的显著变化,但一定程度产生CHG去甲基化变异。由于MSAP试验方法的局限性,关于Osrdr1突变体全基因组DNA胞嘧啶甲基化变异还需更深入研究论证。