1887

Microbiology Society logo

Volume 169, Issue 8

Repurposing the atypical type I-G CRISPR system for bacterial genome engineering Open Access

Abstract

The CRISPR-Cas system functions as a prokaryotic immune system and is highly diverse, with six major types and numerous sub-types. The most abundant are type I CRISPR systems, which utilize a multi-subunit effector, Cascade, and a CRISPR RNA (crRNA) to detect invading DNA species. Detection leads to DNA loading of the Cas3 helicase-nuclease, leading to long-range deletions in the targeted DNA, thus providing immunity against mobile genetic elements (MGE). Here, we focus on the type I-G system, a streamlined, 4-subunit complex with an atypical Cas3 enzyme. We demonstrate that Cas3 helicase activity is not essential for immunity against MGE and explore applications of the Cascade effector for genome engineering in . Long-range, bidirectional deletions were observed when the gene was targeted. Deactivation of the Cas3 helicase activity dramatically altered the types of deletions observed, with small deletions flanked by direct repeats that are suggestive of microhomology mediated end joining. When donor DNA templates were present, both the wild-type and helicase-deficient systems promoted homology-directed repair (HDR), with the latter system providing improvements in editing efficiency, suggesting that a single nick in the target site may promote HDR in using the type I-G system. These findings open the way for further application of the type I-G CRISPR systems in genome engineering.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Cas3 helicase , CRISPR , genome engineering and Type I-G Cascade
Funding
This study was supported by the:
  • China Scholarship Council (Award 202008060345)
    • Principle Award Recipient: QilinShangguan
  • Biotechnology and Biological Sciences Research Council (Award BB/S000313/1)
    • Principle Award Recipient: MalcolmF White
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001373
2023-08-01
2024-05-06
Loading full text...

Full text loading...

/deliver/fulltext/micro/169/8/mic001373.html?itemId=/content/journal/micro/10.1099/mic.0.001373&mimeType=html&fmt=ahah

References

  1. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 2007; 315:1709–1712 [View Article] [PubMed]
     [Google Scholar]
  2. Makarova KS, Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol 2020; 18:67–83 [View Article] [PubMed]
     [Google Scholar]
  3. Barrangou R, Doudna JA. Applications of CRISPR technologies in research and beyond. Nat Biotechnol 2016; 34:933–941 [View Article] [PubMed]
     [Google Scholar]
  4. Cong L, Ran FA, Cox D, Lin S, Barretto R et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339:819–823 [View Article] [PubMed]
     [Google Scholar]
  5. Mali P, Yang L, Esvelt KM, Aach J, Guell M et al. RNA-guided human genome engineering via Cas9. Science 2013; 339:823–826 [View Article] [PubMed]
     [Google Scholar]
  6. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 2015; 163:759–771 [View Article] [PubMed]
     [Google Scholar]
  7. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA et al. An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol 2015; 13:722–736 [View Article] [PubMed]
     [Google Scholar]
  8. Barrangou R. CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip Rev RNA 2013; 4:267–278 [View Article] [PubMed]
     [Google Scholar]
  9. Li Y, Pan S, Zhang Y, Ren M, Feng M et al. Harnessing type I and type III CRISPR-Cas systems for genome editing. Nucleic Acids Res 2016; 44:e34 [View Article] [PubMed]
     [Google Scholar]
  10. Pyne ME, Bruder MR, Moo-Young M, Chung DA, Chou CP. Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium. Sci Rep 2016; 6:25666 [View Article] [PubMed]
     [Google Scholar]
  11. Zhang J, Zong W, Hong W, Zhang Z-T, Wang Y. Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production. Metab Eng 2018; 47:49–59 [View Article] [PubMed]
     [Google Scholar]
  12. Maikova A, Kreis V, Boutserin A, Severinov K, Soutourina O. Using an endogenous CRISPR-Cas system for genome editing in the human pathogen Clostridium difficile. Appl Environ Microbiol 2019; 85:e01416-19 [View Article] [PubMed]
     [Google Scholar]
  13. Cheng F, Gong L, Zhao D, Yang H, Zhou J et al. Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon. J Genet Genomics 2017; 44:541–548 [View Article] [PubMed]
     [Google Scholar]
  14. Csörgő B, León LM, Chau-Ly IJ, Vasquez-Rifo A, Berry JD et al. A compact Cascade-Cas3 system for targeted genome engineering. Nat Methods 2020; 17:1183–1190 [View Article] [PubMed]
     [Google Scholar]
  15. Kiro R, Shitrit D, Qimron U. Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system. RNA Biol 2014; 11:42–44 [View Article] [PubMed]
     [Google Scholar]
  16. Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R et al. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 2014; 5:e00928-13 [View Article] [PubMed]
     [Google Scholar]
  17. Hidalgo-Cantabrana C, Goh YJ, Pan M, Sanozky-Dawes R, Barrangou R. Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus. Proc Natl Acad Sci 2019; 116:15774–15783 [View Article]
     [Google Scholar]
  18. Cañez C, Selle K, Goh YJ, Barrangou R. Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus. FEMS Microbiol Lett 2019; 366:fnz105 [View Article] [PubMed]
     [Google Scholar]
  19. Zhou Y, Yang Y, Li X, Tian D, Ai W et al. Exploiting a conjugative endogenous CRISPR-Cas3 system to tackle multidrug-resistant Klebsiella pneumoniae. EBioMedicine 2023; 88:104445 [View Article] [PubMed]
     [Google Scholar]
  20. Zheng Y, Han J, Wang B, Hu X, Li R et al. Characterization and repurposing of the endogenous Type I-F CRISPR–Cas system of Zymomonas mobilis for genome engineering. Nucleic Acids Res 2019; 47:11461–11475 [View Article]
     [Google Scholar]
  21. Vercoe RB, Chang JT, Dy RL, Taylor C, Gristwood T et al. Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet 2013; 9:e1003454 [View Article] [PubMed]
     [Google Scholar]
  22. Hampton HG, McNeil MB, Paterson TJ, Ney B, Williamson NR et al. CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia. Microbiology 2016; 162:1047–1058 [View Article] [PubMed]
     [Google Scholar]
  23. Xu Z, Li M, Li Y, Cao H, Miao L et al. Native CRISPR-Cas-mediated genome editing enables dissecting and sensitizing clinical multidrug-resistant P. aeruginosa. Cell Rep 2019; 29:1707–1717 [View Article] [PubMed]
     [Google Scholar]
  24. Pan M, Morovic W, Hidalgo-Cantabrana C, Roberts A, Walden KKO et al. Genomic and epigenetic landscapes drive CRISPR-based genome editing in Bifidobacterium. Proc Natl Acad Sci 2022; 119:30 [View Article]
     [Google Scholar]
  25. Xu Z, Li Y, Cao H, Si M, Zhang G et al. A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation. Nucleic Acids Res 2021; 49:16 [View Article] [PubMed]
     [Google Scholar]
  26. Hu C, Ni D, Nam KH, Majumdar S, McLean J et al. Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools. Mol Cell 2022; 82:2754–2768 [View Article] [PubMed]
     [Google Scholar]
  27. Pickar-Oliver A, Black JB, Lewis MM, Mutchnick KJ, Klann TS et al. Targeted transcriptional modulation with type I CRISPR-Cas systems in human cells. Nat Biotechnol 2019; 37:1493–1501 [View Article] [PubMed]
     [Google Scholar]
  28. Tan R, Krueger RK, Gramelspacher MJ, Zhou X, Xiao Y et al. Cas11 enables genome engineering in human cells with compact CRISPR-Cas3 systems. Mol Cell 2022; 82:852–867 [View Article] [PubMed]
     [Google Scholar]
  29. Osakabe K, Wada N, Murakami E, Miyashita N, Osakabe Y. Genome editing in mammalian cells using the CRISPR type I-D nuclease. Nucleic Acids Res 2021; 49:6347–6363 [View Article] [PubMed]
     [Google Scholar]
  30. Dolan AE, Hou Z, Xiao Y, Gramelspacher MJ, Heo J et al. Introducing a spectrum of long-range genomic deletions in human embryonic stem cells using type I CRISPR-cas. Mol Cell 2019; 74:936–950 [View Article] [PubMed]
     [Google Scholar]
  31. Morisaka H, Yoshimi K, Okuzaki Y, Gee P, Kunihiro Y et al. CRISPR-Cas3 induces broad and unidirectional genome editing in human cells. Nat Commun 2019; 10:5302 [View Article] [PubMed]
     [Google Scholar]
  32. Cameron P, Coons MM, Klompe SE, Lied AM, Smith SC et al. Harnessing type I CRISPR-Cas systems for genome engineering in human cells. Nat Biotechnol 2019; 37:1471–1477 [View Article] [PubMed]
     [Google Scholar]
  33. Chen Y, Liu J, Zhi S, Zheng Q, Ma W et al. Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells. Nat Commun 2020; 11: [View Article]
     [Google Scholar]
  34. Mulepati S, Bailey S. In vitro reconstitution of an Escherichia coli RNA-guided immune system reveals unidirectional, ATP-dependent degradation of DNA target. J Biol Chem 2013; 288:22184–22192 [View Article] [PubMed]
     [Google Scholar]
  35. Huo Y, Nam KH, Ding F, Lee H, Wu L et al. Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation. Nat Struct Mol Biol 2014; 21:771–777 [View Article] [PubMed]
     [Google Scholar]
  36. Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P et al. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J 2011; 30:1335–1342 [View Article] [PubMed]
     [Google Scholar]
  37. Redding S, Sternberg SH, Marshall M, Gibb B, Bhat P et al. Surveillance and processing of foreign DNA by the Escherichia coli CRISPR-Cas system. Cell 2015; 163:854–865 [View Article] [PubMed]
     [Google Scholar]
  38. Dillard KE, Brown MW, Johnson NV, Xiao Y, Dolan A et al. Assembly and translocation of a CRISPR-cas primed acquisition complex. Cell 2018; 175:934–946 [View Article] [PubMed]
     [Google Scholar]
  39. Shangguan Q, Graham S, Sundaramoorthy R, White MF. Structure and mechanism of the type I-G CRISPR effector. Nucleic Acids Res 2022; 50:11214–11228 [View Article] [PubMed]
     [Google Scholar]
  40. Westra ER, van Erp PBG, Künne T, Wong SP, Staals RHJ et al. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol Cell 2012; 46:595–605 [View Article] [PubMed]
     [Google Scholar]
  41. Gong B, Shin M, Sun J, Jung C-H, Bolt EL et al. Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3. Proc Natl Acad Sci 2014; 111:16359–16364 [View Article] [PubMed]
     [Google Scholar]
  42. Hochstrasser ML, Taylor DW, Bhat P, Guegler CK, Sternberg SH et al. CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference. Proc Natl Acad Sci 2014; 111:6618–6623 [View Article] [PubMed]
     [Google Scholar]
  43. Xiao Y, Luo M, Dolan AE, Liao M, Ke A. Structure basis for RNA-guided DNA degradation by Cascade and Cas3. Science 2018; 361:eaat0839 [View Article] [PubMed]
     [Google Scholar]
  44. Cui L, Bikard D. Consequences of Cas9 cleavage in the chromosome of Escherichia coli. Nucleic Acids Res 2016; 44:4243–4251 [View Article] [PubMed]
     [Google Scholar]
  45. Chayot R, Montagne B, Mazel D, Ricchetti M. An end-joining repair mechanism in Escherichia coli. Proc Natl Acad Sci 2010; 107:2141–2146 [View Article] [PubMed]
     [Google Scholar]
  46. Hao Y, Wang Q, Li J, Yang S, Zheng Y et al. Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering. Open Biol 2022; 12: [View Article] [PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001373
Loading
Repurposing the atypical type I-G CRISPR system for bacterial genome engineering
Microbiology 169, 001373 (2023); https://doi.org/10.1099/mic.0.001373
/content/journal/micro/10.1099/mic.0.001373
/content/journal/micro/10.1099/mic.0.001373
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error