CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms,but its use in pigs to generate precise genetic modifications has seldom been described.Myostatin(MSTN)is a well-characterized...CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms,but its use in pigs to generate precise genetic modifications has seldom been described.Myostatin(MSTN)is a well-characterized negative regulator of muscle development,and natural mutations in this gene cause a double-muscled phenotype in many species.However,to the best of our knowledge,no naturally occurring mutation in MSTN has been found in pigs.In addition,no living pig models with sophisticated modifications orthologous to natural mutations in MSTN have yet been reported.In this study,we exploited the CRISPR/Cpf1 system to introduce a predefined modification orthologous to the natural MSTN mutation found in Belgian Blue cattle(thus known as the Belgian Blue mutation).Our research demonstrated that the cutting efficiency of CRISPR/Cpf1 was 12.3%in mixed porcine fetal fibroblasts in drug free medium,and 41.7%in clonal colonies obtained using G418 selection.Then,the Cpf1-sgRNA vector,ssODN template,and a self-excision cassette were co-transfected into porcine fetal fibroblasts.After G418 selection,8 clonal colonies were examined and 5 with genetic modification were found.Of these 5,2 harbored the precise 11-bp deletion.Using 1 heterozygous clonal colony,2 cloned Duroc piglets were successfully generated,which was heterozygous for the Belgian Blue mutation.In summary,our results demonstrate that CRISPR/Cpf1 system can be used efficiently to generate double-stranded breaks,and also to mediate homologous recombination to introduce precise genomic modifications in pigs.展开更多
CRISPR/Cas has been coming to prosperity since its discovery and application. It becomes a standard solution for gene editing in the past few years. A guide RNA is used to lead the endonuclease, such as Cas9 and Cpf1,...CRISPR/Cas has been coming to prosperity since its discovery and application. It becomes a standard solution for gene editing in the past few years. A guide RNA is used to lead the endonuclease, such as Cas9 and Cpf1, to specific sites and break the double strand. However, there is also possibility that the system will cut a non-specific position, which is called 'off-target effect'. The off-target cleavage may cause trouble to gene function research or clinic treatment. In order to reveal the target specificity of Cpf1, this study explored the single-nucleotide mismatches by a dual-luciferase system. Our results showed that the poly(T) structure was prohibitive in spacer for Cpf1 targeting. Moreover, rA mismatches seemed to be of the least tolerance for CRISPR/Cpf1, which was same as CRISPR/Cas9. The phenomenon might be attributed to the homology of the two enzymes. In summary, our research suggest that more attention should be paid to off-target effects when using CRISPR/Cpf1 or CRISPR/Cas9, as this is an intrinsic characteristic of the system.展开更多
In the original publication the Supplementary Material and Fig.2 are in correct.The correct versi on of Supplementary Material and Fig.2 are provided in this correction article.The text HBG2 appearing in the article s...In the original publication the Supplementary Material and Fig.2 are in correct.The correct versi on of Supplementary Material and Fig.2 are provided in this correction article.The text HBG2 appearing in the article should be read as HBG1.展开更多
基金supported by the National Transgenic Breeding Program of China (2016ZX08006001)the Doctor’s Fund of Southwest University, China (SWU 118082)
文摘CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms,but its use in pigs to generate precise genetic modifications has seldom been described.Myostatin(MSTN)is a well-characterized negative regulator of muscle development,and natural mutations in this gene cause a double-muscled phenotype in many species.However,to the best of our knowledge,no naturally occurring mutation in MSTN has been found in pigs.In addition,no living pig models with sophisticated modifications orthologous to natural mutations in MSTN have yet been reported.In this study,we exploited the CRISPR/Cpf1 system to introduce a predefined modification orthologous to the natural MSTN mutation found in Belgian Blue cattle(thus known as the Belgian Blue mutation).Our research demonstrated that the cutting efficiency of CRISPR/Cpf1 was 12.3%in mixed porcine fetal fibroblasts in drug free medium,and 41.7%in clonal colonies obtained using G418 selection.Then,the Cpf1-sgRNA vector,ssODN template,and a self-excision cassette were co-transfected into porcine fetal fibroblasts.After G418 selection,8 clonal colonies were examined and 5 with genetic modification were found.Of these 5,2 harbored the precise 11-bp deletion.Using 1 heterozygous clonal colony,2 cloned Duroc piglets were successfully generated,which was heterozygous for the Belgian Blue mutation.In summary,our results demonstrate that CRISPR/Cpf1 system can be used efficiently to generate double-stranded breaks,and also to mediate homologous recombination to introduce precise genomic modifications in pigs.
基金National Natural Science Foundation of China(Grant No.31571403)the Natural Science Foundation of Beijing Municipality(Grant No.2171001)
文摘CRISPR/Cas has been coming to prosperity since its discovery and application. It becomes a standard solution for gene editing in the past few years. A guide RNA is used to lead the endonuclease, such as Cas9 and Cpf1, to specific sites and break the double strand. However, there is also possibility that the system will cut a non-specific position, which is called 'off-target effect'. The off-target cleavage may cause trouble to gene function research or clinic treatment. In order to reveal the target specificity of Cpf1, this study explored the single-nucleotide mismatches by a dual-luciferase system. Our results showed that the poly(T) structure was prohibitive in spacer for Cpf1 targeting. Moreover, rA mismatches seemed to be of the least tolerance for CRISPR/Cpf1, which was same as CRISPR/Cas9. The phenomenon might be attributed to the homology of the two enzymes. In summary, our research suggest that more attention should be paid to off-target effects when using CRISPR/Cpf1 or CRISPR/Cas9, as this is an intrinsic characteristic of the system.
文摘In the original publication the Supplementary Material and Fig.2 are in correct.The correct versi on of Supplementary Material and Fig.2 are provided in this correction article.The text HBG2 appearing in the article should be read as HBG1.
