The authors regret that the grant number“21CJ1402200”in the Acknowledgments session should be replaced as“21JC1402200”.The corrected contents areprovided as follows.
Vogt–Koyanagi–Harada(VKH)disease is a leading cause of blindness in young and middle-aged people.However,the etiology of VKH disease remains unclear.Here,we performed the first trio-based whole-exome sequencing stud...Vogt–Koyanagi–Harada(VKH)disease is a leading cause of blindness in young and middle-aged people.However,the etiology of VKH disease remains unclear.Here,we performed the first trio-based whole-exome sequencing study,which enrolled 25 VKH patients and 50 controls,followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations.A total of 15 de novo mutations in VKH patients were identified,with one of the most important being the membrane palmitoylated protein 2(MPP2)p.K315N(MPP2-N315)mutation.The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions.Additionally,this mutation appears rare,being absent from the 1000 Genome Project and Genome Aggregation Database,and it is highly conserved in 10 species,including humans and mice.Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis(EAU).In vitro,we used clustered regularly interspaced short palindromic repeats(CRISPR‒Cas9)gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315.Levels of cytokines,such as IL-1β,IL-17E,and vascular endothelial growth factor A,were increased,and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells.Mechanistically,the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315,as shown by LC‒MS/MS and Co-IP,and resulted in activation of the ERK3/IL-17E pathway.Overall,our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.展开更多
Genome editing through adeno-associated viral(AAV) vectors is a promising gene therapy strategy for various diseases,especially genetic disorders. However, homologous recombination(HR) efficiency is extremely low in a...Genome editing through adeno-associated viral(AAV) vectors is a promising gene therapy strategy for various diseases,especially genetic disorders. However, homologous recombination(HR) efficiency is extremely low in adult animal models. We assumed that increasing AAV transduction efficiency could increase genome editing activity, especially HR efficiency, for in vivo gene therapy. Firstly, a mouse phenylketonuria(PKU) model carrying a pathogenic R408W mutation in phenylalanine hydroxylase(Pah) was generated. Through co-delivery of the general AAV receptor(AAVR), we found that AAVR could dramatically increase AAV transduction efficiency in vitro and in vivo. Furthermore, co-delivery of SaCas9/sgRNA/donor templates with AAVR via AAV8 vectors increased indel rate over 2-fold and HR rate over 15-fold for the correction of the single mutation in Pah;mice. Moreover, AAVR co-injection successfully increased the site-specific insertion rate of a 1.4 kb Pah cDNA by 11-fold, bringing the HR rate up to 7.3% without detectable global off-target effects. Insertion of Pah cDNA significantly decreased the Phe level and ameliorated PKU symptoms. This study demonstrates a novel strategy to dramatically increase AAV transduction which substantially enhanced in vivo genome editing efficiency in adult animal models, showing clinical potential for both conventional and genome editing-based gene therapy.展开更多
Tensile and isothermal fatigue tests were carried out on an as-rolled Mg-12Gd-3Y-0.5Zr alloy and its heat-treated counterpart at different temperatures. The experimental results show that the ultimate tensile strength...Tensile and isothermal fatigue tests were carried out on an as-rolled Mg-12Gd-3Y-0.5Zr alloy and its heat-treated counterpart at different temperatures. The experimental results show that the ultimate tensile strengths of two alloys decrease very slowly with increasing temperature up to 200℃. The ultimate tensile strength of heat-treated Mg-12Gd-3Y-0.5Zr is slight lower than that of as-rolled counterpart; however, the fatigue strength of heat-treated alloy is higher. The mechanism of fatigue failure was investigated by scanning electron microscopy （SEM） and transmission electron microscopy （TEM）. It shows that cyclic slip combined with environmental effect may be the main crack initiation mechanism.展开更多
CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications.However,it is still infeasible because homologous recombination(HR)is inefficient,especially for nondividing ce...CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications.However,it is still infeasible because homologous recombination(HR)is inefficient,especially for nondividing cells.To overcome the challenge,we report that a homology-independent targeted integration(HITI)strategy is used for permanent integration of high-specificity-activity Factor IX variant(F9 Padua,R338L)at the albumin(Alb)locus in a novel hemophilia B(HB)rat model.The knock-in efficiency reaches 3.66%,as determined by droplet digital PCR(dd PCR).The clotting time is reduced to a normal level four weeks after treatment,and the circulating factor IX(FIX)level is gradually increased up to 52%of the normal level over nine months even after partial hepatectomy,demonstrating the amelioration of hemophilia.Through primer-extension-mediated sequencing(PEM-seq),no significant off-target effect is detected.This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.展开更多
Dear Editor, The clustered regularly interspaced short palindromic repeat （CRISPR） system has been widely adapted to genome editing to either introduce or correct genetic mutations （Wang et al., 2016）. However, du...Dear Editor, The clustered regularly interspaced short palindromic repeat （CRISPR） system has been widely adapted to genome editing to either introduce or correct genetic mutations （Wang et al., 2016）. However, due to competition with the dominant non-homologous end-joining （NHEJ） pathway, precise genome modifications through Cas9-stimulated homologous recombination （HR） is inefficient. Through fusion of cytidine deaminases, APOBEC1 （apolipoprotein B editing complex 1） or AID （activation-induced deaminase）, with Cas9 variants, several groups have developed the cytidine base editor （BE） systems （Komor et al., 2016; Li et al., 2018; Nishida et al., 2016）. The BE system achieves programmable conversion of C-G base pairs to T.A without double-stranded DNA cleavage （Zhou et al., 2017）. More recently, adenine base editors （ABEs）, which efficiently convert A-T base pairs to G-C in genomic DNA, have been developed via fusion of an engineered tRNA adenosine deaminase （ecTadA from Escherichia coh） with nCas9 （Gaudelli et al., 2017）. The ABE system has quickly been adapted to generate disease models and correction of genetic disease in mice （Ryu et al., 2017; Liu et al, 2018）. However, whether the editing efficiency and the targeting scope of ABE could be improved is largely unexplored. In this study, we describe the efficient generation of base-edi- ted mice and rats modeling human diseases through ABEs with highest efficiency up to 100%. We also demonstrate an increase of ABE activity through injection of chemically modified tracrRNA and crRNA in mouse zygotes, and the expansion of editing scope by fusion of an ecTadA mutant to SaCas9n-KKH and Casgn-VQR variants in both cells and embryos. Our study suggests that the ABE system is a powerful and convenient tool to introduce precise base conversions in rodents.展开更多
This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction ...This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.展开更多
基金National Key R&D Program of China(2019YFA0110802 and 2019YFA0802800)the National Natural Science Foundation of China(32025023,31971366)+1 种基金the Shanghai Municipal Commission for Science and Technology(21JC1402200,20140900200)the Innovation Program of Shanghai Municipal Education Commission(2019-01-07-00-05-E00054)。
文摘The authors regret that the grant number“21CJ1402200”in the Acknowledgments session should be replaced as“21JC1402200”.The corrected contents areprovided as follows.
基金We thank the families for participation in this study,and we thank Novogene Technology Co.,Ltd.,for the WES sequencing and analysis.This work was supported by the National Natural Science Foundation Project of China(82070951,82271078)the National Natural Science Foundation Key Program(81930023)+3 种基金The Innovative Research Group Project of Chongqing Education Commission(CXQT19015)the Innovation Supporting Plan of Overseas Study of Chongqing(cx2018010)the National Key Clinical Specialties Construction Program of China,the Chongqing Branch of the National Clinical Research Center for Ocular Diseases,the Chongqing Key Laboratory of Ophthalmology(CSTC,2008CA5003)the Program for Youth Innovation in Future Medicine,Chongqing Medical University(w0047).
文摘Vogt–Koyanagi–Harada(VKH)disease is a leading cause of blindness in young and middle-aged people.However,the etiology of VKH disease remains unclear.Here,we performed the first trio-based whole-exome sequencing study,which enrolled 25 VKH patients and 50 controls,followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations.A total of 15 de novo mutations in VKH patients were identified,with one of the most important being the membrane palmitoylated protein 2(MPP2)p.K315N(MPP2-N315)mutation.The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions.Additionally,this mutation appears rare,being absent from the 1000 Genome Project and Genome Aggregation Database,and it is highly conserved in 10 species,including humans and mice.Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis(EAU).In vitro,we used clustered regularly interspaced short palindromic repeats(CRISPR‒Cas9)gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315.Levels of cytokines,such as IL-1β,IL-17E,and vascular endothelial growth factor A,were increased,and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells.Mechanistically,the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315,as shown by LC‒MS/MS and Co-IP,and resulted in activation of the ERK3/IL-17E pathway.Overall,our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.
基金partially supported by grants from the National Key R&D Program of China (2019YFA0110802)the National Natural Science Foundation of China (81670470 and 81873685)+2 种基金grants from the Shanghai Municipal Commission for Science and Technology (18411953500 and 20140900201)a grant from the Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-05-E00054)the Fundamental Research Funds for the Central Universities
文摘Genome editing through adeno-associated viral(AAV) vectors is a promising gene therapy strategy for various diseases,especially genetic disorders. However, homologous recombination(HR) efficiency is extremely low in adult animal models. We assumed that increasing AAV transduction efficiency could increase genome editing activity, especially HR efficiency, for in vivo gene therapy. Firstly, a mouse phenylketonuria(PKU) model carrying a pathogenic R408W mutation in phenylalanine hydroxylase(Pah) was generated. Through co-delivery of the general AAV receptor(AAVR), we found that AAVR could dramatically increase AAV transduction efficiency in vitro and in vivo. Furthermore, co-delivery of SaCas9/sgRNA/donor templates with AAVR via AAV8 vectors increased indel rate over 2-fold and HR rate over 15-fold for the correction of the single mutation in Pah;mice. Moreover, AAVR co-injection successfully increased the site-specific insertion rate of a 1.4 kb Pah cDNA by 11-fold, bringing the HR rate up to 7.3% without detectable global off-target effects. Insertion of Pah cDNA significantly decreased the Phe level and ameliorated PKU symptoms. This study demonstrates a novel strategy to dramatically increase AAV transduction which substantially enhanced in vivo genome editing efficiency in adult animal models, showing clinical potential for both conventional and genome editing-based gene therapy.
文摘Tensile and isothermal fatigue tests were carried out on an as-rolled Mg-12Gd-3Y-0.5Zr alloy and its heat-treated counterpart at different temperatures. The experimental results show that the ultimate tensile strengths of two alloys decrease very slowly with increasing temperature up to 200℃. The ultimate tensile strength of heat-treated Mg-12Gd-3Y-0.5Zr is slight lower than that of as-rolled counterpart; however, the fatigue strength of heat-treated alloy is higher. The mechanism of fatigue failure was investigated by scanning electron microscopy （SEM） and transmission electron microscopy （TEM）. It shows that cyclic slip combined with environmental effect may be the main crack initiation mechanism.
基金supported by grants from National Key R&D Program of China(2019YFA0110802 and 2019YFA0802800)the National Natural Science Foundation of China(32025023,31971366)+1 种基金grants from the Shanghai Municipal Commission for Science and Technology(21CJ1402200,20140900200)the Innovation Program of Shanghai Municipal Education Commission(2019-01-07-00-05-E00054)。
文摘CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications.However,it is still infeasible because homologous recombination(HR)is inefficient,especially for nondividing cells.To overcome the challenge,we report that a homology-independent targeted integration(HITI)strategy is used for permanent integration of high-specificity-activity Factor IX variant(F9 Padua,R338L)at the albumin(Alb)locus in a novel hemophilia B(HB)rat model.The knock-in efficiency reaches 3.66%,as determined by droplet digital PCR(dd PCR).The clotting time is reduced to a normal level four weeks after treatment,and the circulating factor IX(FIX)level is gradually increased up to 52%of the normal level over nine months even after partial hepatectomy,demonstrating the amelioration of hemophilia.Through primer-extension-mediated sequencing(PEM-seq),no significant off-target effect is detected.This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.
基金This work was partially supported by grants from the National Natural Science Foundation of China （Nos. 81670470 and 81600149）, a grant from the Shanghai Municipal Commission for Science and Technology （14140901600, 18411953500 and 15JC1400201） and a grant from National Key Research and Development Program （2016YFC0905100）. The authors have submitted a patent application （Application Number 2018101425473） based on the results reported in this study.
文摘Dear Editor, The clustered regularly interspaced short palindromic repeat （CRISPR） system has been widely adapted to genome editing to either introduce or correct genetic mutations （Wang et al., 2016）. However, due to competition with the dominant non-homologous end-joining （NHEJ） pathway, precise genome modifications through Cas9-stimulated homologous recombination （HR） is inefficient. Through fusion of cytidine deaminases, APOBEC1 （apolipoprotein B editing complex 1） or AID （activation-induced deaminase）, with Cas9 variants, several groups have developed the cytidine base editor （BE） systems （Komor et al., 2016; Li et al., 2018; Nishida et al., 2016）. The BE system achieves programmable conversion of C-G base pairs to T.A without double-stranded DNA cleavage （Zhou et al., 2017）. More recently, adenine base editors （ABEs）, which efficiently convert A-T base pairs to G-C in genomic DNA, have been developed via fusion of an engineered tRNA adenosine deaminase （ecTadA from Escherichia coh） with nCas9 （Gaudelli et al., 2017）. The ABE system has quickly been adapted to generate disease models and correction of genetic disease in mice （Ryu et al., 2017; Liu et al, 2018）. However, whether the editing efficiency and the targeting scope of ABE could be improved is largely unexplored. In this study, we describe the efficient generation of base-edi- ted mice and rats modeling human diseases through ABEs with highest efficiency up to 100%. We also demonstrate an increase of ABE activity through injection of chemically modified tracrRNA and crRNA in mouse zygotes, and the expansion of editing scope by fusion of an ecTadA mutant to SaCas9n-KKH and Casgn-VQR variants in both cells and embryos. Our study suggests that the ABE system is a powerful and convenient tool to introduce precise base conversions in rodents.
基金This work was partially supported by grants from the National Natural Science Foundation of China (Nos.81670470 and 81600149)a grant from the Shan ghai Municipal Commissi on for Science and Tech nology (17140901600,18411953500 and 15JC1400201)a grant from National Key Research and Development Program (2016YFC0905100).
文摘This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
