CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement.However,the challenge of low editing activity complicates the identification of editing events.In this study,we i...CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement.However,the challenge of low editing activity complicates the identification of editing events.In this study,we introduce multiple single transcript unit surrogate reporter(STU-SR)systems to enhance the selection of genome-edited plants.These systems use the same single guide RNAs designed for endogenous genes to edit reporter genes,establishing a direct link between reporter gene editing activity and that of endogenous genes.Various strategies are used to restore functional reporter genes after genome editing,including efficient single-strand annealing(SSA)for homologous recombination in STUSR-SSA systems.STU-SR-base editor systems leverage base editing to reinstate the start codon,enriching C-to-T and A-to-G base editing events.Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in the monocot rice,encompassing Cas9 nuclease-based targeted mutagenesis,cytosine base editing,and adenine base editing.The systems exhibit compatibility with Cas9 variants,such as the PAM-less SpRY,and are shown to boost genome editing in Brassica oleracea,a dicot vegetable crop.In summary,we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.展开更多
基金supported by the National Key Research and Development Program of China(award no.2023YFD1202900)the National Science Foundation of China(award nos.32270433 and 32101205)+4 种基金the Natural Science Foundation of Sichuan Province(award no.2022NSFSC0143)to Y.Z.and X.T.,the Joint Science and Technology Project between Sichuan Province and Chongqing Municipality(award no.CSTC2021JSCXCYLHX0001)to H.S.and X.T.the Modern Seed Industry Project of Chongqing Municipal Science and Technology Bureau(award no.CSTB2023TIAD-KPX0025)to H.S.the National Science Foundation of China(award no.32301248)to Q.R.the National Science Foundation of China(award no.32072045)to X.Z.supported by the NSF Plant Genome Research Program(award nos.IOS-2029889 and IOS-2132693)to Y.Q.
文摘CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement.However,the challenge of low editing activity complicates the identification of editing events.In this study,we introduce multiple single transcript unit surrogate reporter(STU-SR)systems to enhance the selection of genome-edited plants.These systems use the same single guide RNAs designed for endogenous genes to edit reporter genes,establishing a direct link between reporter gene editing activity and that of endogenous genes.Various strategies are used to restore functional reporter genes after genome editing,including efficient single-strand annealing(SSA)for homologous recombination in STUSR-SSA systems.STU-SR-base editor systems leverage base editing to reinstate the start codon,enriching C-to-T and A-to-G base editing events.Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in the monocot rice,encompassing Cas9 nuclease-based targeted mutagenesis,cytosine base editing,and adenine base editing.The systems exhibit compatibility with Cas9 variants,such as the PAM-less SpRY,and are shown to boost genome editing in Brassica oleracea,a dicot vegetable crop.In summary,we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.
