The clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein(Cas) system has been widely used for genome editing. In this system, the cytosine base editor(CBE) and adenine base edit...The clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein(Cas) system has been widely used for genome editing. In this system, the cytosine base editor(CBE) and adenine base editor(ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine(C) or adenine(A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE(sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal(NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS(F4NLS^(r2)) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE(sCBE) and F4NLS^(r2) involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.展开更多
Base editing,the targeted introduction of point mutations into cellular DNA,holds promise for improving genome-scale functional genome screening to single-nucleotide resolution.Current efforts in prokaryotes,however,r...Base editing,the targeted introduction of point mutations into cellular DNA,holds promise for improving genome-scale functional genome screening to single-nucleotide resolution.Current efforts in prokaryotes,however,remain confined to loss-of-function screens using the premature stop codons-mediated gene inactivation library,which falls far short of fully releasing the potential of base editors.Here,we developed a base editor-mediated functional single nucleotide variant screening pipeline in Escherichia coli.We constructed a library with 31,123 sgRNAs targeting 462 stress response-related genes in E.coli,and screened for adaptive mutations under isobutanol and furfural selective conditions.Guided by the screening results,we successfully identified several known and novel functional mutations.Our pipeline might be expanded to the optimization of other phenotypes or the strain engineering in other microorganisms.展开更多
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic small vessel disease caused by mutations in the NOTCH3 gene. However, the pathogenesis of CADASIL rem...Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic small vessel disease caused by mutations in the NOTCH3 gene. However, the pathogenesis of CADASIL remains unclear, and patients have limited treatment options. Here, we use human induced pluripotent stem cells (hiPSCs) generated from the peripheral blood mononuclear cells of a patient with CADASIL carrying a heterozygous NOTCH3 mutation (c.1261C>T, p.R421C) to develop a disease model. The correction efficiency of different adenine base editors (ABEs) is tested using the HEK293T-NOTCH3 reporter cell line. ABEmax is selected based on its higher efficiency and minimization of predicted off-target effects. Vascular smooth muscle cells (VSMCs) differentiated from CADASIL hiPSCs show NOTCH3 deposition and abnormal actin cytoskeleton structure, and the abnormalities are recovered in corrected hiPSC-derived VSMCs. Furthermore, CADASIL blood vessel organoids generated for in vivo modeling show altered expression of genes related to disease phenotypes, including the downregulation of cell adhesion, extracellular matrix organization, and vessel development. The dual adeno-associated virus (AAV) split-ABEmax system is applied to the genome editing of vascular organoids with an average editing efficiency of 8.82%. Collectively, we present potential genetic therapeutic strategies for patients with CADASIL using blood vessel organoids and the dual AAV split-ABEmax system.展开更多
Single-nucleotide variants account for about half of known pathogenic genetic variants in human.Genome editing strategies by reversing pathogenic point mutations with minimum side effects have great therapeutic potent...Single-nucleotide variants account for about half of known pathogenic genetic variants in human.Genome editing strategies by reversing pathogenic point mutations with minimum side effects have great therapeutic potential and are now being actively pursued.The emerge of precise and effcient genome editing strategies such as base editing and prime editing provide powerful tools for nucleotide conversion without inducing double-stranded DNA breaks(DSBs),which have shown great potential for curing genetic disorders.A diverse toolkit of base editors has been devel-oped to improve the editing effciency and accuracy in different context of application.Here,we summarized the evolving of base editors(BEs),their limitations and future perspective of base editing-based therapeutic strategies.展开更多
Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here,through fusions of different effective cytosine and adenine deaminases wi...Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here,through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore,we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn(D213N)mutation(OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.展开更多
Approximately 140 million people worldwide are homozygous carriers of APOE4(ε4),a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease(AD),91%of whom will develop AD at earlier age tha...Approximately 140 million people worldwide are homozygous carriers of APOE4(ε4),a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease(AD),91%of whom will develop AD at earlier age than heterozygous carriers and noncarriers.Susceptibility to AD could be reduced by targeted editing of APOE4,but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies.Here,we first screened eight cytosine base editor variants at four injection stages(from 1-to 8-cell stage),and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate(up to 100%)with the lowest bystander effects.In particular,80%of AD-susceptibleε4 allele copies were converted to the AD-neutralε3 allele in humanε4-carrying embryos.Stringent control measures combined with targeted deep sequencing,whole genome sequencing,and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells.Furthermore,base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage.Finally,we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia.Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos,a potential approach for reducing human susceptibility to AD or other genetic diseases.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
Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an N...Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an NGG protospacer adjacent motif(PAM),is a widely used nuclease for genome editing in living cells.Cas12a nucleases,targeting T-rich PAMs,have also been recently demonstrated in several plant species.Furthermore,multiple Cas9 and Cas12a engineered variants and orthologs,with different PAM recognition sites,editing efficiencies and fidelity,have been explored in plants.These RNA-guided sequence-specific nucleases(SSN)generate double-stranded breaks(DSBs)in DNA,which trigger non-homologous end-joining(NHEJ)repair or homology-directed repair(HDR),resulting in insertion and deletion(indel)mutations or precise gene replacement,respectively.Alternatively,genome editing can be achieved by base editors without introducing DSBs.So far,several base editors have been applied in plants to introduce C-to-T or A-to-G transitions,but they are still undergoing improvement in editing window size,targeting scope,off-target effects in DNA and RNA,product purity and overall activity.Here,we summarize recent progress on the application of Cas nucleases,engineered Cas variants and base editors in plants.展开更多
Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the convers...Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T(C>T) or A→G(A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing(GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MⅡ transition. Injection of cytosine base editor 3(BE3) mRNA and X-linked Dmdspecific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MⅡ oocytes developed into blastocysts after parthenogenetic activation(PA) or in vitro fertilization(IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4%±2.1%. There 40%–78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five(5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine(9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.展开更多
The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break(DSB)or supplying a DNA donor template for homology-directed repair(HDR)...The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break(DSB)or supplying a DNA donor template for homology-directed repair(HDR).In this study,adenine base editors(ABEs)were used for rapid generation of precise point mutations in two distinct genes,OsWSL5,and OsZEBRA3(Z3),in both rice protoplasts and regenerated plants.The precisely engineered point mutations were stably inherited to subsequent generations.These single nucleotide alterations resulted in single amino acid changes and associated wsl5 and z3 phenotypes as evidenced by white stripe leaf and light green/dark green leaf pattern,respectively.Through selfing and genetic segregation,transgene-free,base edited wsl5 and z3 mutants were obtained in a short period of time.We noticed a novel mutation(V540A)in Z3 locus could also mimic the phenotype of Z3 mutation(S542P).Furthermore,we observed unexpected non-A/G or T/C mutations in the ABE editing window in a few of the edited plants.The ABE vectors and the method from this study could be used to simultaneously generate point mutations in multiple target genes in a single transformation and serve as a useful base editing tool for crop improvement as well as basic studies in plant biology.展开更多
基金supported by the Beijing Scholars Program[BSP041]。
文摘The clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein(Cas) system has been widely used for genome editing. In this system, the cytosine base editor(CBE) and adenine base editor(ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine(C) or adenine(A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE(sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal(NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS(F4NLS^(r2)) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE(sCBE) and F4NLS^(r2) involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.
基金supported by the National Key Research and Development Program of China (2018YFA0901500)the National Natural Science Foundation of China (U2032210)。
文摘Base editing,the targeted introduction of point mutations into cellular DNA,holds promise for improving genome-scale functional genome screening to single-nucleotide resolution.Current efforts in prokaryotes,however,remain confined to loss-of-function screens using the premature stop codons-mediated gene inactivation library,which falls far short of fully releasing the potential of base editors.Here,we developed a base editor-mediated functional single nucleotide variant screening pipeline in Escherichia coli.We constructed a library with 31,123 sgRNAs targeting 462 stress response-related genes in E.coli,and screened for adaptive mutations under isobutanol and furfural selective conditions.Guided by the screening results,we successfully identified several known and novel functional mutations.Our pipeline might be expanded to the optimization of other phenotypes or the strain engineering in other microorganisms.
基金funded by the National Natural Science Foundation of China(31971365)the Guangdong Basic and Applied Basic Research Foundation(2020B1515120090)the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program(2019BT02Y276).
文摘Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic small vessel disease caused by mutations in the NOTCH3 gene. However, the pathogenesis of CADASIL remains unclear, and patients have limited treatment options. Here, we use human induced pluripotent stem cells (hiPSCs) generated from the peripheral blood mononuclear cells of a patient with CADASIL carrying a heterozygous NOTCH3 mutation (c.1261C>T, p.R421C) to develop a disease model. The correction efficiency of different adenine base editors (ABEs) is tested using the HEK293T-NOTCH3 reporter cell line. ABEmax is selected based on its higher efficiency and minimization of predicted off-target effects. Vascular smooth muscle cells (VSMCs) differentiated from CADASIL hiPSCs show NOTCH3 deposition and abnormal actin cytoskeleton structure, and the abnormalities are recovered in corrected hiPSC-derived VSMCs. Furthermore, CADASIL blood vessel organoids generated for in vivo modeling show altered expression of genes related to disease phenotypes, including the downregulation of cell adhesion, extracellular matrix organization, and vessel development. The dual adeno-associated virus (AAV) split-ABEmax system is applied to the genome editing of vascular organoids with an average editing efficiency of 8.82%. Collectively, we present potential genetic therapeutic strategies for patients with CADASIL using blood vessel organoids and the dual AAV split-ABEmax system.
基金supported by CAMS Innovation Fund for Medical Sciences(CIFMS,2022-I2M-1-024,2022-RC180-02 to Z.G.).
文摘Single-nucleotide variants account for about half of known pathogenic genetic variants in human.Genome editing strategies by reversing pathogenic point mutations with minimum side effects have great therapeutic potential and are now being actively pursued.The emerge of precise and effcient genome editing strategies such as base editing and prime editing provide powerful tools for nucleotide conversion without inducing double-stranded DNA breaks(DSBs),which have shown great potential for curing genetic disorders.A diverse toolkit of base editors has been devel-oped to improve the editing effciency and accuracy in different context of application.Here,we summarized the evolving of base editors(BEs),their limitations and future perspective of base editing-based therapeutic strategies.
基金partly funded by the National Natural Science Foundation of China (32188102 to LX)Hainan Yazhou Bay Seed Lab (B21HJ0215 to LX)+2 种基金the Central Public-Interest Scientific Institution-Based Research Fund (S2023ZD03 to LX)Key Laboratory of Gene Editing Technologies (Hainan), ChinaNational Engineering Research Center of Crop Molecular Breeding。
文摘Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here,through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore,we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn(D213N)mutation(OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.
基金supported by Chinese National Science and Technology major project R&D Program of China(2018YFC2000101)Strategic Priority Research Program of Chinese Academy of Science(XDB32060000)+7 种基金National Natural Science Foundation of China(Grant Nos.31871502,31901047,31925016,91957122,82021001,and 31922048)Basic Frontier Scientific Research Program of Chinese Academy of Sciences From 0 to 1 original innovation project(ZDBS-LYSM001)Shanghai Municipal Science and Technology Major Project(2018SHZDZX05)Shanghai City Committee of Science and Technology Project(18411953700,18JC1410100,19XD1424400 and 19YF1455100)Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZDCX20212200 and SHSMU-ZLCX20210200)International Partnership Program of Chinese Academy of Sciences(153D31KYSB20170059)Postdoctoral Science Foundation of China(2020M681417 and 2021T140684)Sailing Program of Shanghai(21YF1453000)(to J.H.).
文摘Approximately 140 million people worldwide are homozygous carriers of APOE4(ε4),a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease(AD),91%of whom will develop AD at earlier age than heterozygous carriers and noncarriers.Susceptibility to AD could be reduced by targeted editing of APOE4,but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies.Here,we first screened eight cytosine base editor variants at four injection stages(from 1-to 8-cell stage),and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate(up to 100%)with the lowest bystander effects.In particular,80%of AD-susceptibleε4 allele copies were converted to the AD-neutralε3 allele in humanε4-carrying embryos.Stringent control measures combined with targeted deep sequencing,whole genome sequencing,and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells.Furthermore,base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage.Finally,we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia.Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos,a potential approach for reducing human susceptibility to AD or other genetic diseases.
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Our plant genome editing research is supported by the National Science Foundation Plant Genome Research Program(IOS-1758745)USDA-NIFA Biotechnology Risk Assessment Research Program(2018-33522-28789)+1 种基金Foundation for Food and Agriculture Research(593603)Syngenta Biotechnology.
文摘Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an NGG protospacer adjacent motif(PAM),is a widely used nuclease for genome editing in living cells.Cas12a nucleases,targeting T-rich PAMs,have also been recently demonstrated in several plant species.Furthermore,multiple Cas9 and Cas12a engineered variants and orthologs,with different PAM recognition sites,editing efficiencies and fidelity,have been explored in plants.These RNA-guided sequence-specific nucleases(SSN)generate double-stranded breaks(DSBs)in DNA,which trigger non-homologous end-joining(NHEJ)repair or homology-directed repair(HDR),resulting in insertion and deletion(indel)mutations or precise gene replacement,respectively.Alternatively,genome editing can be achieved by base editors without introducing DSBs.So far,several base editors have been applied in plants to introduce C-to-T or A-to-G transitions,but they are still undergoing improvement in editing window size,targeting scope,off-target effects in DNA and RNA,product purity and overall activity.Here,we summarize recent progress on the application of Cas nucleases,engineered Cas variants and base editors in plants.
基金supported by the National Key R&D Program of China (2017YFC1001901 and 2017YFA0102801)the National Natural Science Foundation (31671540)+3 种基金the National Transgenic Major Program (2016ZX08006003-006)the Natural Science Foundation of Guangdong Province (2015A020212005 and 2014A030312011)the Key R&D Program of Guangdong Province (2018B020203003)the Guangzhou Science and Technology Project (201803010020)。
文摘Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T(C>T) or A→G(A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing(GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MⅡ transition. Injection of cytosine base editor 3(BE3) mRNA and X-linked Dmdspecific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MⅡ oocytes developed into blastocysts after parthenogenetic activation(PA) or in vitro fertilization(IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4%±2.1%. There 40%–78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five(5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine(9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.
基金This work was supported by National Science Foundation Plant Genome Research Program Grant No.1740874the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project#PEN04659 and Accession#1016432 to Yinong Yang.
文摘The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break(DSB)or supplying a DNA donor template for homology-directed repair(HDR).In this study,adenine base editors(ABEs)were used for rapid generation of precise point mutations in two distinct genes,OsWSL5,and OsZEBRA3(Z3),in both rice protoplasts and regenerated plants.The precisely engineered point mutations were stably inherited to subsequent generations.These single nucleotide alterations resulted in single amino acid changes and associated wsl5 and z3 phenotypes as evidenced by white stripe leaf and light green/dark green leaf pattern,respectively.Through selfing and genetic segregation,transgene-free,base edited wsl5 and z3 mutants were obtained in a short period of time.We noticed a novel mutation(V540A)in Z3 locus could also mimic the phenotype of Z3 mutation(S542P).Furthermore,we observed unexpected non-A/G or T/C mutations in the ABE editing window in a few of the edited plants.The ABE vectors and the method from this study could be used to simultaneously generate point mutations in multiple target genes in a single transformation and serve as a useful base editing tool for crop improvement as well as basic studies in plant biology.