The discovery and utilization of RNA-guided surveillance complexes,such as CRISPR-Cas9,for sequencespecific DNA or RNA cleavage,has revolutionised the process of gene modification or knockdown.To optimise the use of t...The discovery and utilization of RNA-guided surveillance complexes,such as CRISPR-Cas9,for sequencespecific DNA or RNA cleavage,has revolutionised the process of gene modification or knockdown.To optimise the use of this technology,an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements,coupled with high cleavage efficacy and specificity.Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.展开更多
CRISPR-Cas (clustered regularly interspaced short palindromic repeats—CRISPR associated proteins) is a RNA-guided defense immune system that prevents some genetic elements such as plasmids and virus from getting into...CRISPR-Cas (clustered regularly interspaced short palindromic repeats—CRISPR associated proteins) is a RNA-guided defense immune system that prevents some genetic elements such as plasmids and virus from getting into the bacterial cells. Zymomonas mobilis is an ethanologenic bacterium, which encodes a subtype I-F CRISPR-Cas system containing three CRISPR loci and a far distant cas gene cluster. Reverse transcription (RT)-PCR analysis revealed that the CRISPR loci were transcribed on both strands. The Cas proteins were suggested to be expressed based on the previous transcriptomic analysis. Challenging with the invader plasmids containing the artificial protospacer with the protospacer adjacent motif (PAM) of NGG or GG exhibited immune interference activity. However, PAM motif of GG seems more effective than NGG in interference activity. Further, the artificial CRISPR arrays with the spacer sequences targeting to the specific genome sites could also lead to strong immune activity, resulting in almost no transformant grown on the agar plates. It was suggested that bacteria like Z. mobilis ZM4 are lack of the rejoining function to heal the double breakage of genomic DNA made by the CRISPR system. Conclusively, the Type I-F CRISPR-Cas system in Z. mobilis ZM4 is active to functionally defense the invading DNA elements.展开更多
Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestat...Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising cost.Furthermore,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in cattle.In this point of view,electroporation in embryos has been risen as an alternative.Results First,editing efficiency of our electroporation methods were tested for embryos.Presence of mutation on embryo was confirmed by T7E1 assay.With first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,respectively.In case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for BLG.Afterwards,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing.Thirteen recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double KO.Ten surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both genes.Conclusions These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied.Finally,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.展开更多
基金the National Health and Medical Research Council of Australia(Grant No.APP1143008)the Australian Research Council(Grant No.DP180101494)the National Natural Science Foundation of China(Grant No.81772214).
文摘The discovery and utilization of RNA-guided surveillance complexes,such as CRISPR-Cas9,for sequencespecific DNA or RNA cleavage,has revolutionised the process of gene modification or knockdown.To optimise the use of this technology,an exploratory race has ensued to discover or develop new RNA-guided endonucleases with the most flexible sequence targeting requirements,coupled with high cleavage efficacy and specificity.Here we review the constraints of existing gene editing and assess the merits of exploiting the diversity of CRISPR-Cas effectors as a methodology for surmounting these limitations.
文摘CRISPR-Cas (clustered regularly interspaced short palindromic repeats—CRISPR associated proteins) is a RNA-guided defense immune system that prevents some genetic elements such as plasmids and virus from getting into the bacterial cells. Zymomonas mobilis is an ethanologenic bacterium, which encodes a subtype I-F CRISPR-Cas system containing three CRISPR loci and a far distant cas gene cluster. Reverse transcription (RT)-PCR analysis revealed that the CRISPR loci were transcribed on both strands. The Cas proteins were suggested to be expressed based on the previous transcriptomic analysis. Challenging with the invader plasmids containing the artificial protospacer with the protospacer adjacent motif (PAM) of NGG or GG exhibited immune interference activity. However, PAM motif of GG seems more effective than NGG in interference activity. Further, the artificial CRISPR arrays with the spacer sequences targeting to the specific genome sites could also lead to strong immune activity, resulting in almost no transformant grown on the agar plates. It was suggested that bacteria like Z. mobilis ZM4 are lack of the rejoining function to heal the double breakage of genomic DNA made by the CRISPR system. Conclusively, the Type I-F CRISPR-Cas system in Z. mobilis ZM4 is active to functionally defense the invading DNA elements.
基金financially supported by the National Research Foundation of Korea(NRF-2021R1A5A1033157 for SRC program:382 Comparative medicine Disease Research Center,NRF-2021R1F1A105195313)the Research Institute of Veterinary Science,the BK21 Four for Future Veterinary Medicine Leading Education and Research Center,and a Seoul National University(SNU)grant(#550e2020005)。
文摘Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising cost.Furthermore,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in cattle.In this point of view,electroporation in embryos has been risen as an alternative.Results First,editing efficiency of our electroporation methods were tested for embryos.Presence of mutation on embryo was confirmed by T7E1 assay.With first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,respectively.In case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for BLG.Afterwards,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing.Thirteen recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double KO.Ten surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both genes.Conclusions These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied.Finally,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.
