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 wi...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.展开更多
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 stri...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.展开更多
基金partly funded by the National Natural Science Foundation of China (32188102 to LX)Hainan Yazhou Bay Seed Lab (B21HJ0215 to LX)+2 种基金the Central Public-Interest Scientific Institution-Based Research Fund (S2023ZD03 to LX)Key Laboratory of Gene Editing Technologies (Hainan), ChinaNational Engineering Research Center of Crop Molecular Breeding。
文摘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.
基金supported by the STI 2030-Major Projects(2023ZD04074)the National Key Research and Development Program of China(2023YFD1202900)+2 种基金the Nanfan special project of the Chinese Academy of Agricultural Sciences(YBXM2313)the Hainan Seed Industry Laboratory(project of B23CJ0208)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.
文摘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.
