Prime editing(PE)is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates,but is limited by low editing efficiency.We previously fused the M-MLV reverse transcriptase t...Prime editing(PE)is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates,but is limited by low editing efficiency.We previously fused the M-MLV reverse transcriptase to the Cas9 nickase,generating the PE2(v1)system,but the editing efficiency of this system is still low.Here we develop different versions of PE2 by adding the 50-to-30 exonuclease at different positions of the nCas9-M-MLV RT fusion protein.PE2(v2),in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions,deletions,and insertions at several genomic sites by 1.7-to 2.9-fold in plant cells compared to PE2(v1).The improved editing efficiency of PE2(v2)is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1,PE-P2,and PPE systems.Using PE2(v2),we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites.The PE efficiency is further improved by combining PE2(v2)and dualpegRNAs.Taken together,the increased genome editing efficiency of PE2(v2)developed in this study may enhance the applications of PE in plants.展开更多
Blocking the programmed death-ligand 1(PD-L1)on tumor cells with monoclonal antibody therapy has emerged as powerful weapon in cancer immunotherapy.However,only a minority of patients presented immune responses in cli...Blocking the programmed death-ligand 1(PD-L1)on tumor cells with monoclonal antibody therapy has emerged as powerful weapon in cancer immunotherapy.However,only a minority of patients presented immune responses in clinical trials.To develop an alternative treatment method based on immune checkpoint blockade,we designed a novel and efficient CRISPR-Cas9 genome editing system delivered by cationic copolymer aPBAE to downregulate PD-L1 expression on tumor cells via specifically knocking out Cyclin-dependent kinase 5(Cdk5)gene in vivo.The expression of PD-L1 on tumor cells was significantly attenuated by knocking out Cdk5,leading to effective tumor growth inhibition in murine melanoma and lung metastasis suppression in triple-negative breast cancer.Importantly,we demonstrated that aPBAE/Cas9-Cdk5 treatment elicited strong T cell-mediated immune responses in tumor microenvironment that the population of CD8^+T cells was significantly increased while regulatory T cells(Tregs)was decreased.It may be the first case to exhibit direct in vivo PD-L1 downregulation via CRISPR-Cas9 genome editing technology for cancer therapy.It will provide promising strategy for preclinical antitumor treatment through the combination of nanotechnology and genome engineering.展开更多
"The suggestion of the 13^(th) Five-Year Plan regarding the national economy and social development by the central committee of the Communist Party of China" was passed during the 5^(th) plenary session of t..."The suggestion of the 13^(th) Five-Year Plan regarding the national economy and social development by the central committee of the Communist Party of China" was passed during the 5^(th) plenary session of the 18^(th) CPC Central Committee. It proposes new objectives for building a well-off society in an all around way. The objectives inherit the existing achievements for a well-off society by several generations of leaders and enrich the connotation of this theory from different perspectives, such as economy, livelihood, culture, ecology and institutional construction. During the sprint phase of building a well-off society in an all around way, three pairs of relationships should be coordinated, namely the relationship between economic focus and the overall layout in five aspects, between economic development and social justice, and between the international and domestic level.展开更多
基金supported by grants from the National Key Research and Development Program of China(2022YFF1002802)the National Natural Science Foundation of China(32170410)the Science and Technology Innovation Young Talent Team of Shanxi Province(202204051001019).
文摘Prime editing(PE)is a versatile genome editing tool without the need for double-stranded DNA breaks or donor DNA templates,but is limited by low editing efficiency.We previously fused the M-MLV reverse transcriptase to the Cas9 nickase,generating the PE2(v1)system,but the editing efficiency of this system is still low.Here we develop different versions of PE2 by adding the 50-to-30 exonuclease at different positions of the nCas9-M-MLV RT fusion protein.PE2(v2),in which the T5 exonuclease fused to the N-terminus of the nCas9-MMLV fusion protein enhances prime editing efficiency of base substitutions,deletions,and insertions at several genomic sites by 1.7-to 2.9-fold in plant cells compared to PE2(v1).The improved editing efficiency of PE2(v2)is further confirmed by generating increased heritable prime edits in stable transgenic plants compared to the previously established PE-P1,PE-P2,and PPE systems.Using PE2(v2),we generate herbicide-resistant rice by simultaneously introducing mutations causing amino acid substitutions at two target sites.The PE efficiency is further improved by combining PE2(v2)and dualpegRNAs.Taken together,the increased genome editing efficiency of PE2(v2)developed in this study may enhance the applications of PE in plants.
基金supported by the National Natural Science Foundation of China(81872810,81673374 and 81871473)Wuhan University of Science and Technology Plan for Applied Fundamental Research(2017060201010146,China)the Fundamental Research Funds for the Central Universities(2018KFYYXJJ019,2019KFYRCPY049 and 2016YXMS138,China).
文摘Blocking the programmed death-ligand 1(PD-L1)on tumor cells with monoclonal antibody therapy has emerged as powerful weapon in cancer immunotherapy.However,only a minority of patients presented immune responses in clinical trials.To develop an alternative treatment method based on immune checkpoint blockade,we designed a novel and efficient CRISPR-Cas9 genome editing system delivered by cationic copolymer aPBAE to downregulate PD-L1 expression on tumor cells via specifically knocking out Cyclin-dependent kinase 5(Cdk5)gene in vivo.The expression of PD-L1 on tumor cells was significantly attenuated by knocking out Cdk5,leading to effective tumor growth inhibition in murine melanoma and lung metastasis suppression in triple-negative breast cancer.Importantly,we demonstrated that aPBAE/Cas9-Cdk5 treatment elicited strong T cell-mediated immune responses in tumor microenvironment that the population of CD8^+T cells was significantly increased while regulatory T cells(Tregs)was decreased.It may be the first case to exhibit direct in vivo PD-L1 downregulation via CRISPR-Cas9 genome editing technology for cancer therapy.It will provide promising strategy for preclinical antitumor treatment through the combination of nanotechnology and genome engineering.
基金the phased results of national major projects on social science fund in 2014 on"the theory innovation research on a series of important speeches by Xi Jinping,the general secretary"(project item:14&ZDA 002)
文摘"The suggestion of the 13^(th) Five-Year Plan regarding the national economy and social development by the central committee of the Communist Party of China" was passed during the 5^(th) plenary session of the 18^(th) CPC Central Committee. It proposes new objectives for building a well-off society in an all around way. The objectives inherit the existing achievements for a well-off society by several generations of leaders and enrich the connotation of this theory from different perspectives, such as economy, livelihood, culture, ecology and institutional construction. During the sprint phase of building a well-off society in an all around way, three pairs of relationships should be coordinated, namely the relationship between economic focus and the overall layout in five aspects, between economic development and social justice, and between the international and domestic level.
