CRISPR/Cas技术及其在发酵菌株上的应用
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摘要
发酵菌株的改良在发酵工业生产上通常是一项非常重要的内容,而传统的驯化模式周期长、效率低、稳定性差且成本高,很难满足工业化快速生产的要求。规律成簇间隔短回文重复Cas(Clustered Regularly Interspaced Short Palindromic Repeats/Cas,CRISPR/Cas)是细菌及古生菌中的一种适应性免疫系统,在此系统上改进的基因编辑技术能实行RNA导向的DNA精准编辑。因而利用CRISPR/Cas基因编辑技术对菌株的快速构建和优化,是一条快速获得高产菌株的新途径。本文综述了CRISPR/Cas系统组成、工作原理、分类以及该技术在发酵菌株上的应用研究进展,并探索了该技术存在的问题以及目前的解决办法,最后对CRISPR/Cas技术在发酵工程上的潜力进行了展望。
Ferment strains improvement is a vital content in fermentation industry production ordinarily,and it is hard to meet the requirements of rapid industrial production by the screening scheme of traditional domestication because of its time-consuming,inefficiency,instability and high-cost. Clustered Regularly Interspaced Short Palindromic Repeats/Cas(CRISPR/Cas) a gene targeted editing technology improving,an adaptive immune system in bacteria and archabacteria that could edit gene based on this system by DNA accurate edition guided by RNA. Therefore,using CRISPR/Cas gene edition technology rapidly to establish and optimize strains is a novel efficient path to acquire highyielding fermentation strain. In this paper,the CRISPR/Cas components,working principle,classification and its advances in the application on fermentation strain optimization is reviewed. The problems faced by CRISPR/Cas technology are probed,and finally the potential of CRISPR/Cas in fermentation engineering is prospected.
Ferment strains improvement is a vital content in fermentation industry production ordinarily,and it is hard to meet the requirements of rapid industrial production by the screening scheme of traditional domestication because of its time-consuming,inefficiency,instability and high-cost. Clustered Regularly Interspaced Short Palindromic Repeats/Cas(CRISPR/Cas) a gene targeted editing technology improving,an adaptive immune system in bacteria and archabacteria that could edit gene based on this system by DNA accurate edition guided by RNA. Therefore,using CRISPR/Cas gene edition technology rapidly to establish and optimize strains is a novel efficient path to acquire highyielding fermentation strain. In this paper,the CRISPR/Cas components,working principle,classification and its advances in the application on fermentation strain optimization is reviewed. The problems faced by CRISPR/Cas technology are probed,and finally the potential of CRISPR/Cas in fermentation engineering is prospected.
引文
[1]Steensels J,Snoek T,Meersman E,et al.Improving industrial yeast strains:exploiting naturaland artificial diversity[J].Fems Microbiol Rev,2014,38(5):947-995.
[2]王俊明.利用重组大肠杆菌和核糖开关进行L-赖氨酸生产的研究[D].济南:山东大学,2015.
[3]Pourcel C,Salvignol G,Vergnaud G.CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA,and provide additional tools for evolutionary studies[J].Microbiology,2005,151(3):653-663.
[4]Wiedenheft B,Sternberg SH,Doudna JA.RNA-guided genetic silencing systems in bacteria and archaea[J].Nature,2012,482(7385):331-338.
[5]Horvath P,Barrangou R.CRISPR/Cas,the immune system of bacteria and archaea[J].Science,2010,327(5962):167-170.
[6]Bhaya D,Davison M,Barrangou R.CRISPR-Cas systems in bacteria and archaea:versatile small RNAs for adaptive defense and regulation[J].Annu Rev Genet,2011,45(45):273-297.
[7]Liu L,Fan XD.CRISPR-Cas system:a powerful tool for genome engineering[J].Plant Mol Biol,2014,85(3):209-218.
[8]Wright AV,Nunez JK,Doudna JA.Biology and applications of CRISPR systems:harnessing nature's toolbox for genome engineering[J].Cell,2016,164(1-2):29-44.
[9]Barrangou R.Cas9 targeting and the CRISPR revolution[J].Science,2014,344(6185):707-708.
[10]Makarova KS,Haft DH,Barrangou R,et al.Evolution and classification of the CRISPR-Cas systems[J].Nat Rev Microbiol,2011,9(6):467-477.
[11]Carte J,Pfister NT,Compton MM,et al.Binding and cleavage of CRISPR RNA by Cas6[J].Rna,2010,16(11):2181-2188.
[12]Sontheimer E J,Marraffini L A.Microbiology:slicer for DNA[J].Nature,2010,468(7320):45-46.
[13]Makarova KS,Grishin NV,Shabalina SA,et al.A putative RNAinterference-based immune system in prokaryotes:computational analysis of the predicted enzymatic machinery,functional analogies with eukaryotic RNAi,and hypothetical mechanisms of action[J].Biol direct,2006,1(1):7,doi:10.1186/1745-6150-1-7.
[14]Deltcheva E,Chylinski K,Sharma CM,et al.CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III[J].Nature,2011,471(7340):602-607.
[15]Burstein D,Harrington LB,Strutt SC,et al.New CRISPR-Cas systems from uncultivated microbes[J].Nature,2016,542(7640):237-241.
[16]Elcin U.Guardian of genome integrity:cohesin and DNA damage repair[M].United Kingdom:VDM Verlag Dr.Müller,2011:5-93.
[17]Joung JK,Sander JD.TALENs:a widely applicable technology for targeted genome editing[J].Nat Rev Mol Cell Bio,2013,14(1):49-55.
[18]姜泽群,杨烨.CRISPR/Cas技术在生物医药研究中的应用[J].药物生物技术,2016,23(5):412-416.
[19]Lina JG,Giedrius G.Design of a CRISPR-Cas system to increase resistance of Bacillus subtilis to bacteriophage SPP1[J].J Ind Microbiol Biotechnol,2016,43(8):1183-1188.
[20]Wen Z,Minton NP,Zhang Y,et al.Enhanced solvent production by metabolic engineering of a twin-clostridial consortium[J].Metab Eng,2017,39:38-48.
[21]Wang S,Dong S,Wang P,et al.Genome editing in Clostridium saccharoperbutylacetonicum N1-4 using CRISPR-Cas9 system[J].Appl Environ Microbiol,2017,doi:10.1128/AEM.00233-17.
[22]Mark RB,Michael EP,Murray MY,et al.Extending CRISPRCas9 technology from genome editing to transcriptional engineering in the genus Clostridium[J].Appl Environ Microbiol,2016,82(20):6109-6119.
[23]Li Y,Lin Z,Huang C,et al.Metabolic engineering of Escherichia coliusing CRISPR-Cas9 meditated genome editing[J].Metab Eng,2015,31:13-21.
[24]Xia J,Wang L,Zhu JB,et al.Expression of Shewanella frigidimarina fatty acid metabolic genes in E.coli by CRISPR/cas9-coupled lambda red recombineering[J].Biotechnol Lett,2016,38(1):117-122.
[25]Wu J,Du G,Chen J,et al.Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli[J].Sci Rep,2015,5:13477.
[26]Wu J,Zhou P,Zhang X,et al.Efficient de novo synthesis of resveratrol by metabolically engineered Escherichia coli[J].J Ind Microbiol Biotechnol,2017,44(7):1-13.
[27]Li L,Ren YL,Chen JC,et al.Application of CRISPRi for prokaryotic metabolic engineering involving multiple genes,a case study:Controllable P(3HB-co-4HB)biosynthesis[J].Metab Eng,2015,29:160-168.
[28]Cleto S,Jensen JV,Wendisch VF,et al.Corynebacterium glutamicum metabolic engineering with CRISPR interference(CRISPRi)[J].ACS Synth Biol,2016,5(5):375-385.
[29]Heo MJ,Jung HM,Um J,et al.Controlling citrate synthase expression by CRISPR/Cas9 genome editing for n-butanol production in Escherichia coli[J].ACS Synth Biol,2017,6(2):182-189.
[30]Liang L,Liu R,Garst AD,et al.CRISPR En Abled Trackable genome Engineering for isopropanol production in Escherichia coli[J].Metab Eng,2017,41:1-10.
[31]Jakoˇciūnas T,Bonde I,Herrg7)ard M,et al.Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae[J].Metab Eng,2015,28:213-222.
[32]张根林.酿酒酵母中β-香树脂醇合成途径的构建与调控[D].北京:北京理工大学,2015.
[33]Arendt P,Miettinen K,Pollier J,et al.An endoplasmic reticulum-engineered yeast platform for overproduction of triterpenoids[J].Metab Eng,2017,40:165-175.
[34]Amanda AR,Leo D,Maren W,et al.A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae[J].Nucleic Acids Res,2017,45(1):496-508.
[35]Lee YG,Jin YS,Cha YL,et al.Bioethanol production from cellulosic hydrolysates by engineered industrial Saccharomyces cerevisiae[J].Bioresour Technol,2017,228:355-361.
[36]Lian JZ,Zhao H.Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration[J].Biotechnol Bioeng,2016,113(11):2462-2473.
[37]Klein M,Carrillo M,Xiberras J,et al.Towards the exploitation of glycerol's high reducing power in Saccharomyces cerevisiae-based bioprocesses[J].Metab Eng,2016,38:464-472.
[38]Chin YW,Kang WK,Jang HW,et al.CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae[J].J Ind Microbiol Biotechnol,2016,43(11):1517-1525.
[39]Tsai CS,Kong II,Lesmana A,et al.Rapid and marker-free refactoring of xylose-fermenting yeast strains with Cas9/CRISPR[J].Biotechnol Bioeng,2015,112(11):2406-2411.
[40]Dicarlo JE,Norville JE,Mali P,et al.Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems[J].Nucleic Acids Res,2013,41(7):4336-4343.
[41]Kuivanen J,Wang YJ,Richard P.Engineering Aspergillus niger for galactaric acid production:elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9[J].Microb Cell Fact,2016,15(1):210,doi:10.1186/s12934-016-0613-5.
[42]Katayama T,Tanaka Y,Okabe T,et al.Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae[J].Biotechnol Lett,2016,38(4):637-642.
[43]Jacobs JZ,Ciccaglione KM,Tournier V,et al.Implementation of the CRISPR-Cas9 system in fission yeast[J].Nat Commun,2014,5:5344,doi:10.1038/ncomms6344.
[44]Gao Y,Zhao Y.Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing[J].J Integr Plant Biol,2013,56(4):343-349.
[45]Pattanayak V,Lin S,Guilinger JP,et al.High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9nuclease specificity[J].Nat Biotechnol,2013,31(9):839-843.
[46]Lin Y,Cradick TJ,Brown MT,et al.CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences[J].Nucleic Acids Res,2014,42(11):7473-7485.
[47]Klug A.The discovery of zinc fingers and their applications in gene regulation and genome manipulation[J].Annu Rev Biochem,2010,79:213-231.
[48]Li T,Huang S,Zhao X,et al.Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes[J].Nucleic Acids Res,2011,39(14):6315-6325.
[49]Xu K,Ren C,Liu Z,et al.Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus[J].Cell Mol Life Sci,2015,72(2):383-399.
[50]Fu Y,Sander JD,Reyon D,et al.Improving CRISPR-Cas nuclease specificity using truncated guide RNAs[J].Nat Biotechnol,2014,32(3):279-284.
[51]Cho SW,Kim S,Kim Y,et al.Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases[J].Genome Res,2014,24(1):132-141.
[52]Ran FA,Hsu P,Lin CY,et al.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome gditing specificity[J].Cell,2013,154(6):1380-1389.
[2]王俊明.利用重组大肠杆菌和核糖开关进行L-赖氨酸生产的研究[D].济南:山东大学,2015.
[3]Pourcel C,Salvignol G,Vergnaud G.CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA,and provide additional tools for evolutionary studies[J].Microbiology,2005,151(3):653-663.
[4]Wiedenheft B,Sternberg SH,Doudna JA.RNA-guided genetic silencing systems in bacteria and archaea[J].Nature,2012,482(7385):331-338.
[5]Horvath P,Barrangou R.CRISPR/Cas,the immune system of bacteria and archaea[J].Science,2010,327(5962):167-170.
[6]Bhaya D,Davison M,Barrangou R.CRISPR-Cas systems in bacteria and archaea:versatile small RNAs for adaptive defense and regulation[J].Annu Rev Genet,2011,45(45):273-297.
[7]Liu L,Fan XD.CRISPR-Cas system:a powerful tool for genome engineering[J].Plant Mol Biol,2014,85(3):209-218.
[8]Wright AV,Nunez JK,Doudna JA.Biology and applications of CRISPR systems:harnessing nature's toolbox for genome engineering[J].Cell,2016,164(1-2):29-44.
[9]Barrangou R.Cas9 targeting and the CRISPR revolution[J].Science,2014,344(6185):707-708.
[10]Makarova KS,Haft DH,Barrangou R,et al.Evolution and classification of the CRISPR-Cas systems[J].Nat Rev Microbiol,2011,9(6):467-477.
[11]Carte J,Pfister NT,Compton MM,et al.Binding and cleavage of CRISPR RNA by Cas6[J].Rna,2010,16(11):2181-2188.
[12]Sontheimer E J,Marraffini L A.Microbiology:slicer for DNA[J].Nature,2010,468(7320):45-46.
[13]Makarova KS,Grishin NV,Shabalina SA,et al.A putative RNAinterference-based immune system in prokaryotes:computational analysis of the predicted enzymatic machinery,functional analogies with eukaryotic RNAi,and hypothetical mechanisms of action[J].Biol direct,2006,1(1):7,doi:10.1186/1745-6150-1-7.
[14]Deltcheva E,Chylinski K,Sharma CM,et al.CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III[J].Nature,2011,471(7340):602-607.
[15]Burstein D,Harrington LB,Strutt SC,et al.New CRISPR-Cas systems from uncultivated microbes[J].Nature,2016,542(7640):237-241.
[16]Elcin U.Guardian of genome integrity:cohesin and DNA damage repair[M].United Kingdom:VDM Verlag Dr.Müller,2011:5-93.
[17]Joung JK,Sander JD.TALENs:a widely applicable technology for targeted genome editing[J].Nat Rev Mol Cell Bio,2013,14(1):49-55.
[18]姜泽群,杨烨.CRISPR/Cas技术在生物医药研究中的应用[J].药物生物技术,2016,23(5):412-416.
[19]Lina JG,Giedrius G.Design of a CRISPR-Cas system to increase resistance of Bacillus subtilis to bacteriophage SPP1[J].J Ind Microbiol Biotechnol,2016,43(8):1183-1188.
[20]Wen Z,Minton NP,Zhang Y,et al.Enhanced solvent production by metabolic engineering of a twin-clostridial consortium[J].Metab Eng,2017,39:38-48.
[21]Wang S,Dong S,Wang P,et al.Genome editing in Clostridium saccharoperbutylacetonicum N1-4 using CRISPR-Cas9 system[J].Appl Environ Microbiol,2017,doi:10.1128/AEM.00233-17.
[22]Mark RB,Michael EP,Murray MY,et al.Extending CRISPRCas9 technology from genome editing to transcriptional engineering in the genus Clostridium[J].Appl Environ Microbiol,2016,82(20):6109-6119.
[23]Li Y,Lin Z,Huang C,et al.Metabolic engineering of Escherichia coliusing CRISPR-Cas9 meditated genome editing[J].Metab Eng,2015,31:13-21.
[24]Xia J,Wang L,Zhu JB,et al.Expression of Shewanella frigidimarina fatty acid metabolic genes in E.coli by CRISPR/cas9-coupled lambda red recombineering[J].Biotechnol Lett,2016,38(1):117-122.
[25]Wu J,Du G,Chen J,et al.Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli[J].Sci Rep,2015,5:13477.
[26]Wu J,Zhou P,Zhang X,et al.Efficient de novo synthesis of resveratrol by metabolically engineered Escherichia coli[J].J Ind Microbiol Biotechnol,2017,44(7):1-13.
[27]Li L,Ren YL,Chen JC,et al.Application of CRISPRi for prokaryotic metabolic engineering involving multiple genes,a case study:Controllable P(3HB-co-4HB)biosynthesis[J].Metab Eng,2015,29:160-168.
[28]Cleto S,Jensen JV,Wendisch VF,et al.Corynebacterium glutamicum metabolic engineering with CRISPR interference(CRISPRi)[J].ACS Synth Biol,2016,5(5):375-385.
[29]Heo MJ,Jung HM,Um J,et al.Controlling citrate synthase expression by CRISPR/Cas9 genome editing for n-butanol production in Escherichia coli[J].ACS Synth Biol,2017,6(2):182-189.
[30]Liang L,Liu R,Garst AD,et al.CRISPR En Abled Trackable genome Engineering for isopropanol production in Escherichia coli[J].Metab Eng,2017,41:1-10.
[31]Jakoˇciūnas T,Bonde I,Herrg7)ard M,et al.Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae[J].Metab Eng,2015,28:213-222.
[32]张根林.酿酒酵母中β-香树脂醇合成途径的构建与调控[D].北京:北京理工大学,2015.
[33]Arendt P,Miettinen K,Pollier J,et al.An endoplasmic reticulum-engineered yeast platform for overproduction of triterpenoids[J].Metab Eng,2017,40:165-175.
[34]Amanda AR,Leo D,Maren W,et al.A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae[J].Nucleic Acids Res,2017,45(1):496-508.
[35]Lee YG,Jin YS,Cha YL,et al.Bioethanol production from cellulosic hydrolysates by engineered industrial Saccharomyces cerevisiae[J].Bioresour Technol,2017,228:355-361.
[36]Lian JZ,Zhao H.Construction of plasmids with tunable copy numbers in Saccharomyces cerevisiae and their applications in pathway optimization and multiplex genome integration[J].Biotechnol Bioeng,2016,113(11):2462-2473.
[37]Klein M,Carrillo M,Xiberras J,et al.Towards the exploitation of glycerol's high reducing power in Saccharomyces cerevisiae-based bioprocesses[J].Metab Eng,2016,38:464-472.
[38]Chin YW,Kang WK,Jang HW,et al.CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae[J].J Ind Microbiol Biotechnol,2016,43(11):1517-1525.
[39]Tsai CS,Kong II,Lesmana A,et al.Rapid and marker-free refactoring of xylose-fermenting yeast strains with Cas9/CRISPR[J].Biotechnol Bioeng,2015,112(11):2406-2411.
[40]Dicarlo JE,Norville JE,Mali P,et al.Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems[J].Nucleic Acids Res,2013,41(7):4336-4343.
[41]Kuivanen J,Wang YJ,Richard P.Engineering Aspergillus niger for galactaric acid production:elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9[J].Microb Cell Fact,2016,15(1):210,doi:10.1186/s12934-016-0613-5.
[42]Katayama T,Tanaka Y,Okabe T,et al.Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae[J].Biotechnol Lett,2016,38(4):637-642.
[43]Jacobs JZ,Ciccaglione KM,Tournier V,et al.Implementation of the CRISPR-Cas9 system in fission yeast[J].Nat Commun,2014,5:5344,doi:10.1038/ncomms6344.
[44]Gao Y,Zhao Y.Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing[J].J Integr Plant Biol,2013,56(4):343-349.
[45]Pattanayak V,Lin S,Guilinger JP,et al.High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9nuclease specificity[J].Nat Biotechnol,2013,31(9):839-843.
[46]Lin Y,Cradick TJ,Brown MT,et al.CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences[J].Nucleic Acids Res,2014,42(11):7473-7485.
[47]Klug A.The discovery of zinc fingers and their applications in gene regulation and genome manipulation[J].Annu Rev Biochem,2010,79:213-231.
[48]Li T,Huang S,Zhao X,et al.Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes[J].Nucleic Acids Res,2011,39(14):6315-6325.
[49]Xu K,Ren C,Liu Z,et al.Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus[J].Cell Mol Life Sci,2015,72(2):383-399.
[50]Fu Y,Sander JD,Reyon D,et al.Improving CRISPR-Cas nuclease specificity using truncated guide RNAs[J].Nat Biotechnol,2014,32(3):279-284.
[51]Cho SW,Kim S,Kim Y,et al.Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases[J].Genome Res,2014,24(1):132-141.
[52]Ran FA,Hsu P,Lin CY,et al.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome gditing specificity[J].Cell,2013,154(6):1380-1389.