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
Cpf1 is a class 2/type V CRISPR effector that has been recently harnessed for genome editing （Zetsche et al., 2015; Hut et al., 2016; Kim et al., 2016）. Cpff recognizes thymidine-rich sequence as the protospacer-adj...Cpf1 is a class 2/type V CRISPR effector that has been recently harnessed for genome editing （Zetsche et al., 2015; Hut et al., 2016; Kim et al., 2016）. Cpff recognizes thymidine-rich sequence as the protospacer-adjacent motif （PAM） at the 5＇ end of target sequences. In addition, Cpfl requires only a single shorter crRNA and cleaves DNA in a staggered fashion with 5＇ overhangs （Zetsche et al., 2015）.展开更多
The class 2 clustered regularly interspaced short palindromic repeat （CRISPR） systems have been widely used for simultaneous modification of multiple loci in plants. Traditionally, the type II CRISPR-Cas9 or type V ...The class 2 clustered regularly interspaced short palindromic repeat （CRISPR） systems have been widely used for simultaneous modification of multiple loci in plants. Traditionally, the type II CRISPR-Cas9 or type V CRISPR-Cpfl （also known as Cas12a） system is a two-component transcriptional unit （TCTU） in which the Cas9 or Cpf1 protein is expressed from an RNA polymerase （pol） II promoter, whereas the single guide RNA （sgRNA） is typically expressed from a Pol III promoter, such as U6 or U3 promoter.展开更多
CRISPR-Cas technologies have greatly reshaped the biology field.In this review,we discuss the CRISPR-Cas with a particular focus on the associated technologies and applications of CRISPR-Cas9 and CRISPR-Cas12a,which h...CRISPR-Cas technologies have greatly reshaped the biology field.In this review,we discuss the CRISPR-Cas with a particular focus on the associated technologies and applications of CRISPR-Cas9 and CRISPR-Cas12a,which have been most widely studied and used.We discuss the biological mechanisms of CRISPR-Cas as immune defense systems,recently-discovered anti-CRISPR-Cas systems,and the emerging Cas variants(such as xCas9 and Cas13)with unique characteristics.Then,we highlight various CRISPR-Cas biotechnologies,including nucleasedependent genome editing,CRISPR gene regulation(including CRISPR interference/activation),DNA/RNA base editing,and nucleic acid detection.Last,we summarize up-to-date applications of the biotechnologies for synthetic biology and metabolic engineering in various bacterial species.展开更多
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
Bacterial blight(BB), which is caused by Xanthomonas oryzae pv. oryzae(Xoo), is an important rice disease responsible for significant yield losses. In the rice-growing regions of South China where BB outbreaks are com...Bacterial blight(BB), which is caused by Xanthomonas oryzae pv. oryzae(Xoo), is an important rice disease responsible for significant yield losses. In the rice-growing regions of South China where BB outbreaks are common, the resistance of cultivars with BB resistance genes Xa4 and Xa21 has been lost because of rapid changes in the Xoo population structure and virulence. In this study, 421 diverse rice accessions were evaluated regarding their resistance to two Xoo strains, namely GD1358(C5) and IV, which are prevalent pathotypes in South China and overcame the resistance of Xa4 and Xa21, respectively. Using the 4.8 mio filtered SNP dataset, we conducted a genome-wide association study, which identified 13 loci associated with BB resistance, including eight new quantitative trait loci(QTL) and five QTL harboring known BB resistance genes: Xa3/Xa26, xa5, Xa35(t), Xa36(t), Xa40, Xa43(t), and xa44(t). Intriguingly, a steep peak was detected on chromosomes 5 and 11. Six QTL including three new ones, were distributed on chromosome 11, whereas a new QTL q BB5.1 and a known QTL were detected on chromosome 5. Haplotype analyses indicated that the LOC;s05 g01610(Os PRAF2) gene within the q BB5.1 region, which encodes a PRAF protein, is associated with BB resistance. Furthermore, Os PRAF2 knockout lines generated using the CRISPR-Cpf1 system were significantly more resistant to Xoo strains than the wild-type plants. Our results provide researchers and breeders with useful information regarding QTL and gene resources,which may be relevant for developing new BB-resistant rice cultivars.展开更多
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.
基金supported by the National Natural Science Foundation of China(No.3140101312)the Zhejiang Provincial Natural Science Foundation of China(No.LZ14C130003)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences
文摘Cpf1 is a class 2/type V CRISPR effector that has been recently harnessed for genome editing （Zetsche et al., 2015; Hut et al., 2016; Kim et al., 2016）. Cpff recognizes thymidine-rich sequence as the protospacer-adjacent motif （PAM） at the 5＇ end of target sequences. In addition, Cpfl requires only a single shorter crRNA and cleaves DNA in a staggered fashion with 5＇ overhangs （Zetsche et al., 2015）.
文摘The class 2 clustered regularly interspaced short palindromic repeat （CRISPR） systems have been widely used for simultaneous modification of multiple loci in plants. Traditionally, the type II CRISPR-Cas9 or type V CRISPR-Cpfl （also known as Cas12a） system is a two-component transcriptional unit （TCTU） in which the Cas9 or Cpf1 protein is expressed from an RNA polymerase （pol） II promoter, whereas the single guide RNA （sgRNA） is typically expressed from a Pol III promoter, such as U6 or U3 promoter.
基金Natural Science Foundation of Shanghai(Grant No.18ZR1420500)the Science and Technology Commission of Shanghai Municipality(Grant No.18JC1413600).Y.X.acknowledges National 1000 Youth Talents Program.W.L.and Y.Z.are recipients of China Postdoctoral Science Foundation(2018M632119 and 2018M632098).
文摘CRISPR-Cas technologies have greatly reshaped the biology field.In this review,we discuss the CRISPR-Cas with a particular focus on the associated technologies and applications of CRISPR-Cas9 and CRISPR-Cas12a,which have been most widely studied and used.We discuss the biological mechanisms of CRISPR-Cas as immune defense systems,recently-discovered anti-CRISPR-Cas systems,and the emerging Cas variants(such as xCas9 and Cas13)with unique characteristics.Then,we highlight various CRISPR-Cas biotechnologies,including nucleasedependent genome editing,CRISPR gene regulation(including CRISPR interference/activation),DNA/RNA base editing,and nucleic acid detection.Last,we summarize up-to-date applications of the biotechnologies for synthetic biology and metabolic engineering in various bacterial species.
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supported by the National Natural Science Foundation of China(31661143009 and 31571632)the CAAS Innovative Team Awardthe Bill&Melinda Gates Foundation(OPP51587)。
文摘Bacterial blight(BB), which is caused by Xanthomonas oryzae pv. oryzae(Xoo), is an important rice disease responsible for significant yield losses. In the rice-growing regions of South China where BB outbreaks are common, the resistance of cultivars with BB resistance genes Xa4 and Xa21 has been lost because of rapid changes in the Xoo population structure and virulence. In this study, 421 diverse rice accessions were evaluated regarding their resistance to two Xoo strains, namely GD1358(C5) and IV, which are prevalent pathotypes in South China and overcame the resistance of Xa4 and Xa21, respectively. Using the 4.8 mio filtered SNP dataset, we conducted a genome-wide association study, which identified 13 loci associated with BB resistance, including eight new quantitative trait loci(QTL) and five QTL harboring known BB resistance genes: Xa3/Xa26, xa5, Xa35(t), Xa36(t), Xa40, Xa43(t), and xa44(t). Intriguingly, a steep peak was detected on chromosomes 5 and 11. Six QTL including three new ones, were distributed on chromosome 11, whereas a new QTL q BB5.1 and a known QTL were detected on chromosome 5. Haplotype analyses indicated that the LOC;s05 g01610(Os PRAF2) gene within the q BB5.1 region, which encodes a PRAF protein, is associated with BB resistance. Furthermore, Os PRAF2 knockout lines generated using the CRISPR-Cpf1 system were significantly more resistant to Xoo strains than the wild-type plants. Our results provide researchers and breeders with useful information regarding QTL and gene resources,which may be relevant for developing new BB-resistant rice cultivars.
