The accuracy of genomic annotation is crucial for subsequent functional investigations;however,computational protocols used in high-throughput annotation of open reading frames(ORFs)can introduce inconsistencies.These...The accuracy of genomic annotation is crucial for subsequent functional investigations;however,computational protocols used in high-throughput annotation of open reading frames(ORFs)can introduce inconsistencies.These inconsistencies,which lead to non-uniform extension or truncation of sequence ends,pose challenges for downstream analyses.Existing strategies to rectify these inconsistencies are time-consuming and labor-intensive,lacking specific approaches.To address this gap,we developed to GC,a tool that integrates genomic annotation with RNA-seq datasets to rectify annotation inconsistencies.Using to GC,we achieved an accuracy of nearly 100%accuracy in correcting inconsistencies in published Phytophthora sojae ORFs.We applied this innovative pipeline to the GPCR-bigrams gene family,which was predicted to have 42 members in the P.sojae genome but lacked experimental validation.By employing to GC,we identified 32 GPCR-bigram ORFs with inconsistencies between previous annotations and to GC-corrected sequences.Notably,among these were 5 genes(GPCR-TKL9,GPCR-TKL15,GPCR-PDE3,GPCR-AC3,and GPCR-AC4)showed substantial inconsistencies.Experimental gene annotation confirmed the effectiveness of to GC,as sequences obtained through cloning matched those annotated by to GC.Importantly,we discovered two novel GPCRs(GPCR-AC3 and GPCR-AC4),which were previously mispredicted as a single gene.CRISPR/Cas9-mediated knockout experiments revealed the involvement of GPCR-AC4 but not GPCR-AC3 in oospore production,further confirming their status as two separate genes.In addition to P.sojae,the reliability of the to GC pipeline in Phytophthora capsici and Pythium ultimum further emphasizes the robustness of this pipeline.Our findings highlight the utility of to GC for reliable gene model correction,facilitating investigations into biological functions and offering potential applications in diverse species analyses.展开更多
Amyotrophic lateral sclerosis （ALS） is a complex neu- rodegenerative disease with cellular and molecular mechanisms yet to be fully described. Mutations in a number of genes including SOD1 and FUS are associated wit...Amyotrophic lateral sclerosis （ALS） is a complex neu- rodegenerative disease with cellular and molecular mechanisms yet to be fully described. Mutations in a number of genes including SOD1 and FUS are associated with familial ALS. Here we report the generation of induced pluripotent stem cells （iPSCs） from fibroblasts of familial ALS patients bearing SOD1＋1A27~c and FUS＋/GISe6A mutations, respectively. We further gener- ated gene corrected ALS iPSCs using CRISPR/Cas9 system. Genome-wide RNA sequencing （RNA-seq） analysis of motor neurons derived from SOD1＋~A272c and corrected iPSCs revealed 899 aberrant transcripts. Our work may shed light on discovery of early biomarkers and pathways dysregulated in ALS, as well as provide a basis for novel therapeutic strategies to treat ALS.展开更多
Targeted gene integration mediated by CRISPR/Cas9 is a promising therapeutic strategy for monogenic autosomal recessive diseases.In this study,we established a novel all-in-one high-capacity adenovirus(HCAd)that can p...Targeted gene integration mediated by CRISPR/Cas9 is a promising therapeutic strategy for monogenic autosomal recessive diseases.In this study,we established a novel all-in-one high-capacity adenovirus(HCAd)that can pack both CRISPR/Cas9 and donor DNA into the same vector and tested it on a mouse model of mucopolysaccharidosis typeⅦ(MPSⅦ)caused by mutations in theβ-glucuronidase(GUSB)gene.This system allowed targeted integration of promoterless GUSB in the mouse beta-actin gene(m Actb)locus and the co-expression of GUSB with the self-cleaving peptide T2A(T2A)controlled by a strong endogenous m Actb promoter.The in vivo results indicated that the serum GUSB level of MPSⅦmice treated with a single intraperitoneal injection of the HCAd vector achieved 14%of that of wild-type mice,resulting in significant amelioration of lysosomal storage in the liver and spleen.Furthermore,the HCAd was injected intraventricularly in the brain of newborn MPSⅦmice,leading to strongly positive GUSB enzyme staining in the choroid plexus and perivascular spaces of the periventricular regions and reduced lysosome storage.In summary,by using an all-in-one vector,the study provides a universal,one-for-all therapeutic for MPSⅦ,a disease caused by different mutations of the GUSB gene.展开更多
Cases of inherited retinal dystrophy(IRD)can be caused by mutations in the MERTK gene,which results in an autosomal recessive form of blindness(retinitis pigmentosa,RP)characterized by impaired phagocytosis of photore...Cases of inherited retinal dystrophy(IRD)can be caused by mutations in the MERTK gene,which results in an autosomal recessive form of blindness(retinitis pigmentosa,RP)characterized by impaired phagocytosis of photoreceptor outer segments(Pos)by retinal pigment epithelial cells(RPE).Persistent MERTK gene mutations in patient-derived human induced pluripotent stem cells(hiPSCs)pose a challenge for autologous stem cell-derived RPE replacement therapies targeting IRD.展开更多
CRISPR/Cas9 is a versatile genome editing tool that has the potential to be used to cure many genetic diseases.The system works via a guide RNA(gRNA)interacting with the Cas9 protein to form a complex that binds to a ...CRISPR/Cas9 is a versatile genome editing tool that has the potential to be used to cure many genetic diseases.The system works via a guide RNA(gRNA)interacting with the Cas9 protein to form a complex that binds to a specific DNA sequence.1 The site-specific DNA binding feature of the Cas9 system can be utilized in a variety of ways to correct gene mutations or to regulate gene expression.First,the Cas9 protein can make a site-specific double-stranded break that is mainly repaired by homology-directed repair or non-homologous end-joining.展开更多
Genetic manipulation of human pluripotent stem cells(hPSCs)provides a powerful tool for modeling diseases and developing future medicine.Recently a number of independent genome-editing techniques were developed,includ...Genetic manipulation of human pluripotent stem cells(hPSCs)provides a powerful tool for modeling diseases and developing future medicine.Recently a number of independent genome-editing techniques were developed,including plasmid,bacterial artificial chromosome,adeno-associated virus vector,zinc finger nuclease,transcription activator-like effecter nuclease,and helper-dependent adenoviral vector.Gene editing has been successfully employed in different aspects of stem cell research such as gene correction,mutation knock-in,and establishment of reporter cell lines(Raya et al.,2009;Howden et al.,2011;Li et al.,2011;Liu et al.,2011b;Papapetrou et al.,2011;Sebastiano et al.,2011;Soldner et al.,2011;Zou et al.,2011a).These techniques combined with the utility of hPSCs will significantly influence the area of regenerative medicine.展开更多
基金supported by the grants to Min Qiu and Ming Wang from the National Natural Science Foundation of China(32100160 and 32100044)the grants to Ming Wang from the Jiangsu“Innovative and Entrepreneurial Talent”Program,China(JSSCRC2021510)the grants to Yuanchao Wang from the Chinese Modern Agricultural Industry Technology System(CARS-004-PS14)。
文摘The accuracy of genomic annotation is crucial for subsequent functional investigations;however,computational protocols used in high-throughput annotation of open reading frames(ORFs)can introduce inconsistencies.These inconsistencies,which lead to non-uniform extension or truncation of sequence ends,pose challenges for downstream analyses.Existing strategies to rectify these inconsistencies are time-consuming and labor-intensive,lacking specific approaches.To address this gap,we developed to GC,a tool that integrates genomic annotation with RNA-seq datasets to rectify annotation inconsistencies.Using to GC,we achieved an accuracy of nearly 100%accuracy in correcting inconsistencies in published Phytophthora sojae ORFs.We applied this innovative pipeline to the GPCR-bigrams gene family,which was predicted to have 42 members in the P.sojae genome but lacked experimental validation.By employing to GC,we identified 32 GPCR-bigram ORFs with inconsistencies between previous annotations and to GC-corrected sequences.Notably,among these were 5 genes(GPCR-TKL9,GPCR-TKL15,GPCR-PDE3,GPCR-AC3,and GPCR-AC4)showed substantial inconsistencies.Experimental gene annotation confirmed the effectiveness of to GC,as sequences obtained through cloning matched those annotated by to GC.Importantly,we discovered two novel GPCRs(GPCR-AC3 and GPCR-AC4),which were previously mispredicted as a single gene.CRISPR/Cas9-mediated knockout experiments revealed the involvement of GPCR-AC4 but not GPCR-AC3 in oospore production,further confirming their status as two separate genes.In addition to P.sojae,the reliability of the to GC pipeline in Phytophthora capsici and Pythium ultimum further emphasizes the robustness of this pipeline.Our findings highlight the utility of to GC for reliable gene model correction,facilitating investigations into biological functions and offering potential applications in diverse species analyses.
文摘Amyotrophic lateral sclerosis （ALS） is a complex neu- rodegenerative disease with cellular and molecular mechanisms yet to be fully described. Mutations in a number of genes including SOD1 and FUS are associated with familial ALS. Here we report the generation of induced pluripotent stem cells （iPSCs） from fibroblasts of familial ALS patients bearing SOD1＋1A27~c and FUS＋/GISe6A mutations, respectively. We further gener- ated gene corrected ALS iPSCs using CRISPR/Cas9 system. Genome-wide RNA sequencing （RNA-seq） analysis of motor neurons derived from SOD1＋~A272c and corrected iPSCs revealed 899 aberrant transcripts. Our work may shed light on discovery of early biomarkers and pathways dysregulated in ALS, as well as provide a basis for novel therapeutic strategies to treat ALS.
基金supported by the National Natural Science Foundation of China(81471772,81773265,31601086)the Key Research and Development Plan of Shaanxi Province(2018SF-106)。
文摘Targeted gene integration mediated by CRISPR/Cas9 is a promising therapeutic strategy for monogenic autosomal recessive diseases.In this study,we established a novel all-in-one high-capacity adenovirus(HCAd)that can pack both CRISPR/Cas9 and donor DNA into the same vector and tested it on a mouse model of mucopolysaccharidosis typeⅦ(MPSⅦ)caused by mutations in theβ-glucuronidase(GUSB)gene.This system allowed targeted integration of promoterless GUSB in the mouse beta-actin gene(m Actb)locus and the co-expression of GUSB with the self-cleaving peptide T2A(T2A)controlled by a strong endogenous m Actb promoter.The in vivo results indicated that the serum GUSB level of MPSⅦmice treated with a single intraperitoneal injection of the HCAd vector achieved 14%of that of wild-type mice,resulting in significant amelioration of lysosomal storage in the liver and spleen.Furthermore,the HCAd was injected intraventricularly in the brain of newborn MPSⅦmice,leading to strongly positive GUSB enzyme staining in the choroid plexus and perivascular spaces of the periventricular regions and reduced lysosome storage.In summary,by using an all-in-one vector,the study provides a universal,one-for-all therapeutic for MPSⅦ,a disease caused by different mutations of the GUSB gene.
基金supported by the Norway Grants and Technology Agency of the Czech Republic(KAPPA project No.TO01000107)European Regional Development Fund by Programme Johannes Amos Comenius(No.CZ.02.01.01/00/22_008/0004562 Project"Excellence in Regenerative Medicine")+4 种基金funded by Generalitat Valenciana and co-financed with ERDF funds(OP ERDF of Comunitat Valenciana 2014-2020)supported by funds from Foundation AFM-Telethon(21180)Fundacion Marato TV3 with Ref:202010-10 and CIAICO/2021/115supported by funds from Instituto de Salud Carlos III(ISCIII)of the Spanish Ministry of Health(PI20/01119 to D.L.co-funded by European Regional Development Fund[FEDER funds]"A Way to Make Europe")Univerdidad CEU Cardenal Herrera grant GIR 23/04.
文摘Cases of inherited retinal dystrophy(IRD)can be caused by mutations in the MERTK gene,which results in an autosomal recessive form of blindness(retinitis pigmentosa,RP)characterized by impaired phagocytosis of photoreceptor outer segments(Pos)by retinal pigment epithelial cells(RPE).Persistent MERTK gene mutations in patient-derived human induced pluripotent stem cells(hiPSCs)pose a challenge for autologous stem cell-derived RPE replacement therapies targeting IRD.
基金supported by the New Frontiers in Research Fund(NFRF),administered by the Social Sciences and Humanities Research Council(SSHRC)on behalf of CIHR,NSERC,and SSHRC(Grant#NFRFE-2021-00713).
文摘CRISPR/Cas9 is a versatile genome editing tool that has the potential to be used to cure many genetic diseases.The system works via a guide RNA(gRNA)interacting with the Cas9 protein to form a complex that binds to a specific DNA sequence.1 The site-specific DNA binding feature of the Cas9 system can be utilized in a variety of ways to correct gene mutations or to regulate gene expression.First,the Cas9 protein can make a site-specific double-stranded break that is mainly repaired by homology-directed repair or non-homologous end-joining.
文摘Genetic manipulation of human pluripotent stem cells(hPSCs)provides a powerful tool for modeling diseases and developing future medicine.Recently a number of independent genome-editing techniques were developed,including plasmid,bacterial artificial chromosome,adeno-associated virus vector,zinc finger nuclease,transcription activator-like effecter nuclease,and helper-dependent adenoviral vector.Gene editing has been successfully employed in different aspects of stem cell research such as gene correction,mutation knock-in,and establishment of reporter cell lines(Raya et al.,2009;Howden et al.,2011;Li et al.,2011;Liu et al.,2011b;Papapetrou et al.,2011;Sebastiano et al.,2011;Soldner et al.,2011;Zou et al.,2011a).These techniques combined with the utility of hPSCs will significantly influence the area of regenerative medicine.
