Skip to main content
SpringerLink
Log in

CRISPR–Cas9/CRISPRi tools for cell factory construction in E. coli

  • Review
  • Published:
World Journal of Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The innovative CRISPR–Cas based genome editing technology provides some functionality and advantages such as the high efficiency and specificity as well as ease of handling. Both aspects of the CRISPR–Cas9 system including genetic engineering and gene regulation are advantageously applicable to the construction of microbial cell factories. As one of the most extensively used cell factories, E. coli has been engineered to produce various high value-added chemical compounds such as pharmaceuticals, biochemicals, and biofuels. Therefore, to improve the production of valuable metabolites, many investigations have been performed by focusing on CRISPR–Cas- based metabolic engineering of this host. In the current review, the biology underlying CRISPR–Cas9 system was briefly explained and then the applications of CRISPR–Cas9/CRISPRi tools were considered for cell factory construction in E. coli.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Abdelaal AS, Yazdani SS. (2020). Development and use of CRISPR in industrial applications. In: Genome Engineering via CRISPR-Cas9 System. Elsevier, pp 177–197

  • Adiego-Pérez B, Randazzo P, Daran JM, Verwaal R, Roubos JA, Daran-Lapujade P, Van Der Oost J. (2019). Multiplex genome editing of microorganisms using CRISPR-Cas FEMS microbiology letters 366:fnz086

  • Alkhnbashi OS, Meier T, Mitrofanov A, Backofen R, Voß B (2020) CRISPR-Cas Bioinformatics Methods 172:3–11

    CAS  PubMed  Google Scholar 

  • An J, Zhang W, Jing X, Nie Y, Xu Y. (2020). Reconstitution of TCA cycle involving l-isoleucine dioxygenase for hydroxylation of l-isoleucine in Escherichia coli using CRISPR-Cas9 3 Biotech 10:1–10

  • Bae S, Park J, Kim J-S. (2014). Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases Bioinformatics 30:1473–1475

  • Bhushan K. (2020). Evolution and molecular mechanism of CRISPR/Cas9 systems. In: Genome Engineering via CRISPR-Cas9 System. Elsevier, pp 15–25

  • Boch J et al (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509–1512

    CAS  PubMed  Google Scholar 

  • Bortesi L, Fischer R. (2015). The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology advances, 33: 41–52

  • Carroll D (2011) Genome engineering with zinc-finger nucleases. Genetics 188:773–782

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chaikind B, Van Rossum HM, Miller A, Perkovich P, Szyjka S, Patel K. (2020). Applications of CRISPRI in high throughput metabolic engineering. Google Patents

  • Charpentier E, Elsholz A, Marchfelder A. (2019). CRISPR-Cas: more than ten years and still full of mysteries. Taylor & Francis

  • Chen S, Yao Y, Zhang Y, Fan G. (2020). CRISPR system: Discovery, development and off-target detection Cellular Signalling, 109577

  • Cho S, Choe D, Lee E, Kim SC, Palsson B, Cho B-K. (2018). High-level dCas9 expression induces abnormal cell morphology in Escherichia coli ACS synthetic biology 7:1085–1094

  • Cho S, Shin J, Cho B-K (2018) Applications of CRISPR/Cas system to bacterial metabolic engineering. Int J Mol Sci 19:1089

    PubMed Central  Google Scholar 

  • Choudhary M, Joshi S, Singh P, Srivastava N. (2020). Biofuel production from lignocellulosic biomass: Introduction and metabolic engineering for fermentation scale-up. In: Genetic and Metabolic Engineering for Improved Biofuel Production from Lignocellulosic Biomass. Elsevier, pp 1–12

  • Chuai G-h, Wang Q-L, Liu Q (2017) In silico meets in vivo: towards computational CRISPR-based sgRNA design. Trends Biotechnol 35:12–21

    CAS  PubMed  Google Scholar 

  • Cress BF, Leitz QD, Kim DC, Amore TD, Suzuki JY, Linhardt RJ, Koffas MA. (2017). CRISPRi-mediated metabolic engineering of E. coli for O-methylated anthocyanin production Microbial cell factories 16:10

  • Cress BF et al. (2015). CRISPathBrick: modular combinatorial assembly of type II-A CRISPR arrays for dCas9-mediated multiplex transcriptional repression in E. coli. ACS Syn Biol 4:987–1000

  • Das M, Patra P, Ghosh A (2020) Metabolic engineering for enhancing microbial biosynthesis of advanced biofuels. Renew Sustain Energy Rev 119:109562

    CAS  Google Scholar 

  • Dasgupta A, Chowdhury N, De RK (2020) Metabolic pathway engineering: perspectives and applications. Comput Methods Programs Biomed 192:105436

    PubMed  Google Scholar 

  • Deyell M, Ameta S, Nghe P. (2019). Large scale control and programming of gene expression using CRISPR. In: Seminars in cell & developmental biology, Elsevier

  • Ding W, Weng H, Du G, Chen J, Kang Z. (2017). 5-Aminolevulinic acid production from inexpensive glucose by engineering the C4 pathway in Escherichia coli. J Indus Microbiol Biotechnol 44:1127–1135

  • Doench JG et al. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol, 34:184

  • Dong C. (2019). Programming bacterial gene expression using synthetic CRISPR-Cas transcriptional regulators.

  • Dong X et al (2020) CRISPRi-guided multiplexed fine-tuning of metabolic flux for enhanced Lacto-N-neotetraose production in Bacillus subtilis. J Agricul Food Chem 68:2477–2484

    CAS  Google Scholar 

  • Ebrahimi V, Hashemi A. (2020). Challenges of in vitro genome editing with CRISPR/Cas9 and possible solutions: a review Gene, 144813

  • Fokum E et al. (2019). Metabolic engineering of bacterial strains using CRISPR/Cas9 systems for biosynthesis of value-added products. Food Biosci, 28:125–132

  • Fujiwara R, Noda S, Tanaka T, Kondo A. (2020). Metabolic engineering of Escherichia coli for shikimate pathway derivative production from glucose–xylose co-substrate Nature Communications, 11:1–12

  • Geng Y, Deng Z, Sun Y. (2016). An insight into the protospacer adjacent motif of Streptococcus pyogenes Cas9 with artificially stimulated RNA-guided-Cas9 DNA cleavage flexibility. RSC Adv 6:33514–33522

  • Guo T, Xin Y, Zhang Y, Gu X, Kong J (2019) A rapid and versatile tool for genomic engineering in Lactococcus lactis. Microb Cell Fact 18:22

    PubMed  PubMed Central  Google Scholar 

  • Hashemi A (2018) CRISPR-Cas system as a genome engineering platform: applications in biomedicine and biotechnology. Curr Gene Ther 18:115–124

    CAS  PubMed  Google Scholar 

  • Heo M-J, Jung H-M, Um J, Lee S-W, Oh M-K. (2017). Controlling citrate synthase expression by CRISPR/Cas9 genome editing for n-butanol production in Escherichia coli. ACS Synth Biol 6:182–189

  • Herai RH. (2019). Avoiding the off-target effects of CRISPR/cas9 system is still a challenging accomplishment for genetic transformation. Gene 700:176–178

  • Hille F, Richter H, Wong SP, Bratovič M, Ressel S, Charpentier E (2018) The biology of CRISPR-Cas: backward and forward. Cell 172:1239–1259

    CAS  PubMed  Google Scholar 

  • Hsu PD et al. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 31:827

  • Huminiecki L, Horbańczuk J (2018) The functional genomic studies of resveratrol in respect to its anti-cancer effects. Biotechnol Adv 36:1699–1708

    CAS  PubMed  Google Scholar 

  • Ibrahim A, ÖZSÖZ M SZ, Tirah G, Gideon O (2019) Genome engineering using the CRISPR Cas9 system. J Biomed Pharm Sci, 2:2

  • Kim SK, Han GH, Seong W, Kim H, Kim S-W, Lee D-H, Lee S-G (2016) CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production. Metab Eng 38:228–240

    CAS  PubMed  Google Scholar 

  • Kim SK, Seong W, Han GH, Lee D-H, Lee S-G (2017) CRISPR interference-guided multiplex repression of endogenous competing pathway genes for redirecting metabolic flux in Escherichia coli. Microb Cell Fact 16:188

    PubMed  PubMed Central  Google Scholar 

  • Koonin EV, Makarova KS (2019) Origins and evolution of CRISPR-Cas systems. Philosoph Trans Royal Soc B 374:20180087

    CAS  Google Scholar 

  • Li D, Zhou H, Zeng X. (2019). Battling CRISPR-Cas9 off-target genome editing. Springer

  • Li Q-S, Li Y, Deora GS, Ruan B-F (2019) Derivatives and analogues of resveratrol: recent advances in structural modification. Mini Rev Med Chem 19:809–825

    CAS  PubMed  Google Scholar 

  • Li Q, Seys FM, Minton NP, Yang J, Jiang Y, Jiang W, Yang S. (2019) CRISPR–Cas9D10A nickase‐assisted base editing in the solvent producer Clostridium beijerinckii. Biotechnol Bioeng, 116:1475–1483

  • Li S, Jendresen CB, Grünberger A, Ronda C, Jensen SI, Noack S, Nielsen AT (2016) Enhanced protein and biochemical production using CRISPRi-based growth switches. Metab Eng 38:274–284

    CAS  PubMed  Google Scholar 

  • Li Y et al. (2015). Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing. Metabolic Eng, 31:13–21

  • Liang L, Liu R, Garst AD, Lee T, Beckham GT, Gill RT (2017) CRISPR EnAbled trackable genome engineering for isopropanol production in Escherichia coli. Metab Eng 41:1–10

    CAS  PubMed  Google Scholar 

  • Liu H, Wei Z, Dominguez A, Li Y, Wang X, Qi LS. (2015). CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation. Bioinformatics, 31:3676–3678

  • Liu M, Rehman S, Tang X, Gu K, Fan Q, Chen D, Ma W (2019) Methodologies for improving HDR efficiency. Front Genet 9:691

    PubMed  PubMed Central  Google Scholar 

  • Lunge A, Choudhary E, Sharma R, Gupta R, Agarwal N. (2020). Functional understanding of CRISPR interference: its advantages and limitations for gene silencing in bacteria. In: Genome Engineering via CRISPR-Cas9 System. Elsevier, pp 199–218

  • Lv L, Ren Y-L, Chen J-C, Wu Q, Chen G-Q. (2015). Application of CRISPRi for prokaryotic metabolic engineering involving multiple genes, a case study: controllable P (3HB-co-4HB) biosynthesis. Metabol Eng, 29:160–168

  • Ma M, Ye AY, Zheng W, Kong L. (2013). A guide RNA sequence design platform for the CRISPR/Cas9 system for model organism genomes. BioMed Res Int 2013

  • Macpherson CR, Scherf A (2015) Flexible guide-RNA design for CRISPR applications using protospacer workbench. Nat Biotechnol 33:805

    CAS  PubMed  Google Scholar 

  • Makarova KS et al. (2015). An updated evolutionary classification of CRISPR–Cas systems. Nat Rev Microbiol, 13:722–736

  • Marcellin E, Nielsen LK (2018) Advances in analytical tools for high throughput strain engineering. Curr Opin Biotechnol 54:33–40

    CAS  PubMed  Google Scholar 

  • McCarty NS, Graham AE, Studená L, Ledesma-Amaro R (2020) Multiplexed CRISPR technologies for gene editing and transcriptional regulation Nature. Communications 11:1–13

    Google Scholar 

  • Mitsui R, Yamada R, Ogino H (2019) CRISPR system in the yeast Saccharomyces cerevisiae and its application in the bioproduction of useful chemicals. World J Microbiol Biotechnol 35:111

    PubMed  Google Scholar 

  • Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E. (2014). CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res, 42:W401-W407

  • Moon SB, Ko J-H, Kim Y-S (2019) Recent advances in the CRISPR genome editing tool set. Exp Mol Med 51:1–11

    CAS  PubMed  Google Scholar 

  • Mougiakos I, Bosma EF, Ganguly J, van der Oost J, van Kranenburg R (2018) Hijacking CRISPR-Cas for high-throughput bacterial metabolic engineering: advances and prospects. Curr Opin Biotechnol 50:146–157

    CAS  PubMed  Google Scholar 

  • Naito Y, Hino K, Bono H, Ui-Tei K. (2015). CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics, 31:1120–1123

  • Nandy D, Maity A, Mitra AK (2020) Target-specific gene delivery in plant systems and their expression: Insights into recent developments. J Biosci 45:30

    CAS  PubMed  Google Scholar 

  • Nishimasu H et al. (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 156:935–949

  • O’Brien A, Bailey TL. (2014). GT-Scan: identifying unique genomic targets Bioinformatics 30:2673–2675

  • Pan S, Reed JL (2018) Advances in gap-filling genome-scale metabolic models and model-driven experiments lead to novel metabolic discoveries. Curr Opin Biotechnol 51:103–108

    CAS  PubMed  Google Scholar 

  • Pawelczak KS, Gavande NS, VanderVere-Carozza PS, Turchi JJ (2018) Modulating DNA repair pathways to improve precision genome engineering. ACS Chem Biol 13:389–396

    CAS  PubMed  Google Scholar 

  • Prykhozhij SV, Vinothkumar Rajan DG, Berman JN. (2015). CRISPR multitargeter: a web tool to find common and unique CRISPR single guide RNA targets in a set of similar sequences, PloS one, 10

  • Reisch CR, Prather KL. (2015). The no-SCAR (S carless C as9 A ssisted R ecombineering) system for genome editing in Escherichia coli. Sci Rep, 5:1–12

  • Satowa D et al. (2020). Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl‐CoA supply. Biotechnol Bioeng

  • Schultenkämper K, Brito LF, Wendisch VF. (2020). Impact of CRISPR interference on strain development in biotechnology. Biotechnol Appl Biochem

  • Shen C-C, Hsu M-N, Chang C-W, Lin M-W, Hwu J-R, Tu Y, Hu Y-C. (2019). Synthetic switch to minimize CRISPR off-target effects by self-restricting Cas9 transcription and translation. Nucleic Acids Res, 47:e13-e13

  • Shmakov S et al. (2015). Discovery and functional characterization of diverse class 2 CRISPR-Cas systems, Mol cell, 60:385–397

  • Singh V. (2020). An introduction to genome editing CRISPR-Cas systems. In: Genome Engineering via CRISPR-Cas9 System. Elsevier, pp 1–13

  • Sledzinski P, Nowaczyk M, Olejniczak M (2020) Computational tools and resources supporting CRISPR-Cas. Exp Cell 9:1288

    Google Scholar 

  • Song M. (2017). The CRISPR/Cas9 system: Their delivery, in vivo and ex vivo applications and clinical development by startups. Biotechnol Progress, 33:1035–1045

  • Song X, Huang H, Xiong Z, Ai L, Yang S. (2017). CRISPR-Cas9D10A nickase-assisted genome editing in Lactobacillus casei. Appl Environ Microbiol, 83:e01259–01217

  • Standage-Beier K, Zhang Q, Wang X (2015) Targeted large-scale deletion of bacterial genomes using CRISPR-nickases. ACS Synth Biol 4:1217–1225

    CAS  PubMed  PubMed Central  Google Scholar 

  • Stemmer M, Thumberger T, del Sol Keyer M, Wittbrodt J, Mateo JL. (2015). CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool, PloS one, 10

  • Terns RM, Terns MP (2014) CRISPR-based technologies: prokaryotic defense weapons repurposed. Trends Genet 30:111–118

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tian J, Yang G, Gu Y, Sun X, Lu Y, Jiang W. (2020). Developing an endogenous quorum-sensing based CRISPRi circuit for autonomous and tunable dynamic regulation of multiple targets in industrial Streptomyces bioRxiv

  • Tian P, Wang J, Shen X, Rey JF, Yuan Q, Yan Y (2017) Fundamental CRISPR-Cas9 tools and current applications in microbial systems. Synth Sys Biotechnolo 2:219–225

    Google Scholar 

  • Trimidal SG et al (2019) Can designer indels be tailored by gene editing? can indels be customized? BioEssays 41:1900126

    Google Scholar 

  • Van Der Oost J, Westra ER, Jackson RN, Wiedenheft B (2014) Unravelling the structural and mechanistic basis of CRISPR–Cas systems. Nat Rev Microbiol 12:479–492

    PubMed  PubMed Central  Google Scholar 

  • Vento JM, Crook N, Beisel CL (2019) Barriers to genome editing with CRISPR in bacteria. J Indus Microbiol Biotechnol 46:1327–1341

    CAS  Google Scholar 

  • Vigouroux A, Bikard D. (2020). CRISPR tools to control gene expression in bacteria. Microbiol Mol Biol Rev, 84

  • Wang B, Guo Y, Xu Z, Tu R, Wang Q. (2020). Genomic, transcriptomic, and metabolic characterizations of Escherichia coli adapted to branched-chain higher alcohol tolerance. Appl Microbiol Biotechnol, 1–14

  • Wang Q, Zeng W, Zhou J (2019) Effect of gene knock-out of L-tyrosine transport system on L-tyrosine production in Escherichia coli Sheng wu gong cheng xue bao. Chin J Biotechnol 35:1247–1255

    CAS  Google Scholar 

  • Wilson LO, O’Brien AR, Bauer DC (2018) The current state and future of CRISPR-Cas9 gRNA design tools. Front Pharmacol 9:749

    PubMed  PubMed Central  Google Scholar 

  • Wong N, Liu W, Wang X (2015) WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system. Genome Biol 16:218

    PubMed  PubMed Central  Google Scholar 

  • Wright AV, Nuñez JK, Doudna JA (2016) Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164:29–44

    CAS  PubMed  Google Scholar 

  • Wu J, Du G, Chen J, Zhou J (2015) Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli. Sci Rep 5:13477

    PubMed  PubMed Central  Google Scholar 

  • Wu J, Zhang X, Zhu Y, Tan Q, He J, Dong M (2017) Rational modular design of metabolic network for efficient production of plant polyphenol pinosylvin. Sci Rep 7:1–15

    Google Scholar 

  • Wu J, Zhou P, Zhang X, Dong M (2017) efficient de novo synthesis of resveratrol by metabolically engineered Escherichia coli. J Indus Microbiol Biotechnol 44:1083–1095

    CAS  Google Scholar 

  • Wu M-Y, Sung L-Y, Li H, Huang C-H, Hu Y-C (2017) Combining CRISPR and CRISPRi systems for metabolic engineering of E. coli and 1, 4-BDO biosynthesis. ACS Synth Biol 6:2350–2361

    CAS  PubMed  Google Scholar 

  • Wu Y et al (2020) Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis. Nucleic Acids Res 48:996–1009

    PubMed  Google Scholar 

  • Xia J et al (2016) Expression of Shewanella frigidimarina fatty acid metabolic genes in E. coli by CRISPR/cas9-coupled lambda Red recombineering. Biotechnol let 38:117–122

    CAS  Google Scholar 

  • Xie S, Shen B, Zhang C, Huang X, Zhang Y. (2014). sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites. PloS one, 9

  • Xu H et al (2015) Sequence determinants of improved CRISPR sgRNA design. Genome Res 25:1147–1157

    CAS  PubMed  PubMed Central  Google Scholar 

  • Xu S, Wang Q, Zeng W, Li Y, Shi G, Zhou J. (2020). Construction of a heat-inducible Escherichia coli strain for efficient de novo biosynthesis of L-tyrosine. Process Biochem

  • Yin H et al (2018) Partial DNA-guided Cas9 enables genome editing with reduced off-target activity. Nat Chemical Biol 14:311

    CAS  Google Scholar 

  • Zhan T, Rindtorff N, Betge J. (2019). Ebert MP, Boutros M CRISPR/Cas9 for cancer research and therapy. In: Seminars in cancer biology, Elsevier, pp 106–119

  • Zhang B, Zhang X-M, Wang W, Liu Z-Q, Zheng Y-G (2019) Metabolic engineering of Escherichia coli for d-pantothenic acid production. Food Chem 294:267–275

    CAS  PubMed  Google Scholar 

  • Zhang H, Cheng Q-X, Liu A-M, Zhao G-P, Wang J (2017) A novel and efficient method for bacteria genome editing employing both CRISPR/Cas9 and an antibiotic resistance cassette. Front Microbiol 8:812

    PubMed  PubMed Central  Google Scholar 

  • Zhu LJ, Holmes BR, Aronin N, Brodsky MH. (2014). CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems, PloS one, 9

  • Ziegler M, Takors R (2020) Reduced and minimal cell factories in bioprocesses: towards a streamlined chassis. Minimal Cells: Design. Construction, Biotechnological Applications. Springer, pp 1–44

    Google Scholar 

  • Zou X et al (2020) Pathway construction and metabolic engineering for fermentative production of β-alanine in Escherichia coli. Appl Microbiol Biotechnol 104:2545–2559

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the research deputy of Shahid Beheshti University of Medical Sciences in Tehran, Iran.

Funding

Not applicable.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, No. 2660, Vali-e-Asr Ave, Tehran, Iran

    Atieh Hashemi

Authors
  1. Atieh Hashemi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Atieh Hashemi.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hashemi, A. CRISPR–Cas9/CRISPRi tools for cell factory construction in E. coli. World J Microbiol Biotechnol 36, 96 (2020). https://doi.org/10.1007/s11274-020-02872-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11274-020-02872-9

Keywords

Search

Navigation