β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A〉G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-th...β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A〉G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A〉G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A〉G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A〉G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we consb'ucted nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes.Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.展开更多
Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease- causing mutations in patients. However, problems such as mosaic...Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease- causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical applicaUon of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high- fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.展开更多
Hepatitis B virus(HBV) is an important human pathogen belonging to the Hepadnaviridae family, Orthohepadnavirus genus. Over 240 million people are infected with HBV worldwide. The reverse transcription during its geno...Hepatitis B virus(HBV) is an important human pathogen belonging to the Hepadnaviridae family, Orthohepadnavirus genus. Over 240 million people are infected with HBV worldwide. The reverse transcription during its genome replication leads to low fidelity DNA synthesis, which is the source of variability in the viral proteins. To investigate the variability quantitatively, we retrieved amino acid sequences of 5,167 records of all available HBV genotypes(A–J) from the Genbank database. The amino acid sequences encoded by the open reading frames(ORF) S/C/P/X in the HBV genome were extracted and subjected to alignment. We analyzed the variability of the lengths and the sequences of proteins as well as the frequencies of amino acids. It comprehensively characterized the variability and conservation of HBV proteins at the level of amino acids. Especially for the structural proteins, hepatitis B surface antigens(HBsAg), there are potential sites critical for virus assembly and immune recognition. Interestingly, the preS1 domains in HBsAg were variable at some positions of amino acid residues, which provides a potential mechanism of immune-escape for HBV, while the preS2 and S domains were conserved in the lengths of protein sequences. In the S domain, the cysteine residues and the secondary structures of the alpha-helix and beta-sheet were likely critical for the stable folding of all HBsAg components. Also, the preC domain and C-terminal domain of the core protein are highly conserved. However, the polymerases(HBpol) and the HBx were highly variable at the amino acid level. Our research provides a basis for understanding the conserved and important domains of HBV viral proteins, which could be potential targets for anti-virus therapy.展开更多
Grass carp(Ctenopharyngodon idella) is one of the most important species in China.Decabromodiphenyl ethane(DBDPE) is a brominated flame retardant that has been used widely in industry, and has been observed to acc...Grass carp(Ctenopharyngodon idella) is one of the most important species in China.Decabromodiphenyl ethane(DBDPE) is a brominated flame retardant that has been used widely in industry, and has been observed to accumulate in the tissues of fish from South China. Evidence has shown that DBDPE is toxic to aquatic animals, but the molecular response has been unclear. MicroRNAs(miRNAs) are small noncoding and negative regulatory RNAs that are 20–24 nucleotides in length, which are involved in a wide range of biological processes. We took advantage of deep-sequencing techniques to accurately and comprehensively profile the kidney mi RNA expression of grass carp after 8 weeks of oral exposure to DBDPE. After mapping sequencing data to the genome and Expressed Sequence Tags(ESTs) of grass carp, we identified 493 miRNAs in the sequenced grass carp samples, which included 51 new miRNAs. The results indicated that 5 miRNAs were significantly down-regulated and 36 miRNAs were significantly up-regulated(FDR 〈 0.001,1.5-fold change) after DBDPE exposure. Real-time quantitative PCR(RT-qPCR) was performed on 4 miRNAs from the two samples, and the sequencing and RT-qPCR data were consistent. This study provides the first comprehensive identification of grass carp miRNAs, and the first expression analysis of grass carp miRNAs following DBDPE exposure.The results indicated that miRNAs have potential for use as biomarkers.展开更多
Understanding the regulatory networks for germ cell fate specification is necessary to developing strategies for improving the efficiency of germ cell production in vitro.In this study,we developed a coupled screening...Understanding the regulatory networks for germ cell fate specification is necessary to developing strategies for improving the efficiency of germ cell production in vitro.In this study,we developed a coupled screening strategy that took advantage of an arrayed bi-molecular fluorescence complementation(BiFC)platform for protein-protein interaction screens and epiblast-like cell(EpiLC)-induction assays using reporter mouse embryonic stem cells(mESCs).Investigation of candidate interaction partners of core human pluripotent factors OCT4,NANOG,KLF4 and SOX2 in EpiLC differentiation assays identified novel primordial germ cell(PGC)-inducing factors including BEN-domain(BEND/Bend)family members.Through RNA-seq,ChIP-seq,and ATAC-seq analyses,we showed that Bend5 worked together with Bend4 and helped mark chromatin boundaries to promote EpiLC induction in vitro.Our findings suggest that BEND/Bend proteins represent a new family of transcriptional modulators and chromatin boundary factors that participate in gene expression regulation during early germline development.展开更多
Dear Editor, Many human genetic diseases are caused by pathogenic single nucleotide mutations. Animal models are often used to study these diseases where the pathogenic point mutations are created and/or corrected thr...Dear Editor, Many human genetic diseases are caused by pathogenic single nucleotide mutations. Animal models are often used to study these diseases where the pathogenic point mutations are created and/or corrected through gene editing (e.g., the CRISPP-JCas9 system) (Komor et al., 2017; Liang et al., 2017). CRISPR/Cas9-mediated gene editing depends on DNA double-strand breaks (DSBs), which can be of low efficiency and lead to indels and off-target cleavage (Kim et al., 2016). We and others have shown that base editors (BEs) may represent an attractive alternative for disease mouse model generation (Liang et al., 2017; Kim et al., 2017). Compared to CRISPR/ Cas9, cytidine base editors (CBEs) can generate C·G to T·A mutations in mouse zygotes without activating DSB repair pathways (Liang et al., 2017; Kim et al., 2017; Komor et al., 2016). In addition, CBEs showed much lower off-targets than CRISPR]Cas9 (Kim et al., 2017), making the editing process potentially safer and more controllable. Recently, adenine base editors (ABEs) that were developed from the tRNA- specific adenosine deaminase (TADA) of Escherichia coli were also reported (Gaudelli et al., 2017). As a RNA-guided programmable adenine deaminase, ABE can catalyze the conversion of A to I. Following DNA replication, base I is replaced by G, resulting in A·T to G·C conversion (Gaudelli et al., 2017; Hu et al., 2018). The development of ABEs has clearly expanded the editing capacity and application of BEs. Here, we tested whether ABEs could effectively generate disease mouse models, and found high efficiency by ABEs in producing edited mouse zygotes and mice with single-nucleotide substitutions.展开更多
Bivalves are species-rich mollusks with prominent protective roles in coastal ecosystems.Across these ancient lineages,colony-founding larvae anchor themselves either by byssus production or by cemented attachment.The...Bivalves are species-rich mollusks with prominent protective roles in coastal ecosystems.Across these ancient lineages,colony-founding larvae anchor themselves either by byssus production or by cemented attachment.The latter mode of sessile life is strongly molded by left-right shell asymmetry during larval development of Ostreoida oysters such as Crassostrea hongkongensis.Here,we sequenced the genome of C.hongkongensis in high resolution and compared it to reference bivalve genomes to unveil genomic determinants driving cemented attachment and shell asymmetry.Importantly,loss of the homeobox gene Antennapedia(Antp)and broad expansion of lineagespecific extracellular gene families are implicated in a shift from byssal to cemented attachment in bivalves.Comparative transcriptomic analysis shows a conspicuous divergence between leftright asymmetrical C.hongkongensis and symmetrical Pinctada fucata in their expression profiles.Especially,a couple of orthologous transcription factor genes and lineage-specific shell-related gene families including that encoding tyrosinases are elevated,and may cooperatively govern asymmetrical shell formation in Ostreoida oysters.展开更多
基金We are grateful to Dr. Qi Zhou for helpful suggestions. This work was supported by National Key R&D Program of China (2017YFC1001901 and 2017YFC1001600), the Science and Technology Planning Project of Guangdong Province (2015B020228002), the Guangzhou Science and Technology Project (201707010085) and the National Natural Science Foundation of China (Grant No. 81771579).
文摘β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A〉G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A〉G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A〉G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A〉G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we consb'ucted nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes.Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.
基金This work was supported by the National Natural Science Foundation of China (Grant Nos. 91640119, 31601196, 81330055, 31371508, and 31671540), the Natural Science Foundation of Guangdong Province (2016A030310206 and 2014A030312011), the Science and Technology Planning Project of Guangdong Province (2015B020228002 and 2015A020212005), the Guangzhou Science and Technology Project (201605030012 and 201707010085), and the Fundamental Research Funds for the Central Universities (161gzd13 and 161gpy31). We would also like to acknowledge the support of CA211653, CPRIT RP160462, the Welch Foundation Q-1673, and the C-BASS Shared Resource at the Dan L. Duncan Cancer Center (DLDCC) of Baylor College of Medicine (P30CA125123).
文摘Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease- causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical applicaUon of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high- fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.
基金partially supported by the National Natural Science Foundation of China (Nos. U1611265, 81773271 and 31672536)the Key Projects of Department of Education of Guangdong Province (No. 2017KZDXM088)
文摘Hepatitis B virus(HBV) is an important human pathogen belonging to the Hepadnaviridae family, Orthohepadnavirus genus. Over 240 million people are infected with HBV worldwide. The reverse transcription during its genome replication leads to low fidelity DNA synthesis, which is the source of variability in the viral proteins. To investigate the variability quantitatively, we retrieved amino acid sequences of 5,167 records of all available HBV genotypes(A–J) from the Genbank database. The amino acid sequences encoded by the open reading frames(ORF) S/C/P/X in the HBV genome were extracted and subjected to alignment. We analyzed the variability of the lengths and the sequences of proteins as well as the frequencies of amino acids. It comprehensively characterized the variability and conservation of HBV proteins at the level of amino acids. Especially for the structural proteins, hepatitis B surface antigens(HBsAg), there are potential sites critical for virus assembly and immune recognition. Interestingly, the preS1 domains in HBsAg were variable at some positions of amino acid residues, which provides a potential mechanism of immune-escape for HBV, while the preS2 and S domains were conserved in the lengths of protein sequences. In the S domain, the cysteine residues and the secondary structures of the alpha-helix and beta-sheet were likely critical for the stable folding of all HBsAg components. Also, the preC domain and C-terminal domain of the core protein are highly conserved. However, the polymerases(HBpol) and the HBx were highly variable at the amino acid level. Our research provides a basis for understanding the conserved and important domains of HBV viral proteins, which could be potential targets for anti-virus therapy.
基金financially supported by a General Project granted by the National Natural Science Foundation of China (No. 51178002)
文摘Grass carp(Ctenopharyngodon idella) is one of the most important species in China.Decabromodiphenyl ethane(DBDPE) is a brominated flame retardant that has been used widely in industry, and has been observed to accumulate in the tissues of fish from South China. Evidence has shown that DBDPE is toxic to aquatic animals, but the molecular response has been unclear. MicroRNAs(miRNAs) are small noncoding and negative regulatory RNAs that are 20–24 nucleotides in length, which are involved in a wide range of biological processes. We took advantage of deep-sequencing techniques to accurately and comprehensively profile the kidney mi RNA expression of grass carp after 8 weeks of oral exposure to DBDPE. After mapping sequencing data to the genome and Expressed Sequence Tags(ESTs) of grass carp, we identified 493 miRNAs in the sequenced grass carp samples, which included 51 new miRNAs. The results indicated that 5 miRNAs were significantly down-regulated and 36 miRNAs were significantly up-regulated(FDR 〈 0.001,1.5-fold change) after DBDPE exposure. Real-time quantitative PCR(RT-qPCR) was performed on 4 miRNAs from the two samples, and the sequencing and RT-qPCR data were consistent. This study provides the first comprehensive identification of grass carp miRNAs, and the first expression analysis of grass carp miRNAs following DBDPE exposure.The results indicated that miRNAs have potential for use as biomarkers.
基金the National Key R&D Program of China(2017YFA0102801)The National Natural Science Foundation of China(Grant Nos.31930058,31671540,32170802,and 31301082)+1 种基金Natural Science Foundation of Guangdong Province(2015B020228002,2017A030313093)Guangdong Basic and Applied Basic Research Foundation(2019A1515011422,2021A1515010759).
文摘Understanding the regulatory networks for germ cell fate specification is necessary to developing strategies for improving the efficiency of germ cell production in vitro.In this study,we developed a coupled screening strategy that took advantage of an arrayed bi-molecular fluorescence complementation(BiFC)platform for protein-protein interaction screens and epiblast-like cell(EpiLC)-induction assays using reporter mouse embryonic stem cells(mESCs).Investigation of candidate interaction partners of core human pluripotent factors OCT4,NANOG,KLF4 and SOX2 in EpiLC differentiation assays identified novel primordial germ cell(PGC)-inducing factors including BEN-domain(BEND/Bend)family members.Through RNA-seq,ChIP-seq,and ATAC-seq analyses,we showed that Bend5 worked together with Bend4 and helped mark chromatin boundaries to promote EpiLC induction in vitro.Our findings suggest that BEND/Bend proteins represent a new family of transcriptional modulators and chromatin boundary factors that participate in gene expression regulation during early germline development.
文摘Dear Editor, Many human genetic diseases are caused by pathogenic single nucleotide mutations. Animal models are often used to study these diseases where the pathogenic point mutations are created and/or corrected through gene editing (e.g., the CRISPP-JCas9 system) (Komor et al., 2017; Liang et al., 2017). CRISPR/Cas9-mediated gene editing depends on DNA double-strand breaks (DSBs), which can be of low efficiency and lead to indels and off-target cleavage (Kim et al., 2016). We and others have shown that base editors (BEs) may represent an attractive alternative for disease mouse model generation (Liang et al., 2017; Kim et al., 2017). Compared to CRISPR/ Cas9, cytidine base editors (CBEs) can generate C·G to T·A mutations in mouse zygotes without activating DSB repair pathways (Liang et al., 2017; Kim et al., 2017; Komor et al., 2016). In addition, CBEs showed much lower off-targets than CRISPR]Cas9 (Kim et al., 2017), making the editing process potentially safer and more controllable. Recently, adenine base editors (ABEs) that were developed from the tRNA- specific adenosine deaminase (TADA) of Escherichia coli were also reported (Gaudelli et al., 2017). As a RNA-guided programmable adenine deaminase, ABE can catalyze the conversion of A to I. Following DNA replication, base I is replaced by G, resulting in A·T to G·C conversion (Gaudelli et al., 2017; Hu et al., 2018). The development of ABEs has clearly expanded the editing capacity and application of BEs. Here, we tested whether ABEs could effectively generate disease mouse models, and found high efficiency by ABEs in producing edited mouse zygotes and mice with single-nucleotide substitutions.
基金support from the Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou),China(Grant No. GML2019ZD0407)the Key Deployment Project of Centre for Ocean Mega-Research of Science, Chinese Academy of Science (Grant No. COMS2019Q11)+6 种基金the National Natural Science Foundation of China (Grant Nos. 32073002 and 31902404)the China Agricultural Research System (Grant No. CARS-49)the Science and Technology Program of Guangzhou, China (Grant No. 201804020073)Natural Science Foundation of Guangdong, China (Grant No. 2020A1515011533)the Program of the Pearl River Young Talents of Science and Technology in Guangzhou of China (Grant No. 201806010003)the Institution of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences (Grant Nos. ISEE2018PY01, ISEE2018PY03, and ISEE2018ZD01)the Science and Technology Planning Project of Guangdong Province, China (Grant Nos. 2017B030314052 and 201707010177)
文摘Bivalves are species-rich mollusks with prominent protective roles in coastal ecosystems.Across these ancient lineages,colony-founding larvae anchor themselves either by byssus production or by cemented attachment.The latter mode of sessile life is strongly molded by left-right shell asymmetry during larval development of Ostreoida oysters such as Crassostrea hongkongensis.Here,we sequenced the genome of C.hongkongensis in high resolution and compared it to reference bivalve genomes to unveil genomic determinants driving cemented attachment and shell asymmetry.Importantly,loss of the homeobox gene Antennapedia(Antp)and broad expansion of lineagespecific extracellular gene families are implicated in a shift from byssal to cemented attachment in bivalves.Comparative transcriptomic analysis shows a conspicuous divergence between leftright asymmetrical C.hongkongensis and symmetrical Pinctada fucata in their expression profiles.Especially,a couple of orthologous transcription factor genes and lineage-specific shell-related gene families including that encoding tyrosinases are elevated,and may cooperatively govern asymmetrical shell formation in Ostreoida oysters.