CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering

Abstract

Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn7-like transposon from Vibrio cholerae, which uses a Type I-F CRISPR–Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA–assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Streamlined single-plasmid system for RNA-guided DNA integration.
Fig. 2: INTEGRATE supports high-efficiency insertion of large (10-kb) genetic payloads.
Fig. 3: Orthogonal INTEGRATE systems facilitate multiple, iterative insertions.
Fig. 4: Multi-spacer CRISPR arrays direct single-step multiplexed insertions.
Fig. 5: Robust and highly accurate INTEGRATE activity in additional Gram-negative bacteria.

Data availability

NGS data are available in the NCBI Sequence Read Archive (BioProject accession code PRJNA668381). Published genomes used for analyses were obtained from the NCBI (accessions codes CP001509.3, U00096.3, CP009273.1 and AE015451.2). Datasets generated and analyzed in the current study, as well as custom scripts used for the described data analyses, are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

Custom Python scripts used for the described NGS data analyses are available online via GitHub (https://github.com/sternberglab/Vo_etal_2020). The INTEGRATE guide RNA design tool and associated documentation are available online via GitHub (https://github.com/sternberglab/INTEGRATE-guide-RNA-tool).

References

  1. 1.

    Dunbar, C. E. et al. Gene therapy comes of age. Science 359, eaan4672 (2018).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  2. 2.

    Gelvin, S. B. Integration of Agrobacterium T-DNA into the plant genome. Annu. Rev. Genet. 51, 195–217 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    Davy, A. M., Kildegaard, H. F. & Andersen, M. R. Cell factory engineering. Cell Syst. 4, 262–275 (2017).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. 4.

    Brophy, J. A. N. et al. Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria. Nat. Microbiol. 3, 1043–1053 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  5. 5.

    Miyazaki, R. & van der Meer, J. R. A new large-DNA-fragment delivery system based on integrase activity from an integrative and conjugative element. Appl. Environ. Microbiol. 79, 4440–4447 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Martínez-García, E. & de Lorenzo, V. Transposon-based and plasmid-based genetic tools for editing genomes of gram-negative bacteria. Methods Mol. Biol. 813, 267–283 (2012).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  7. 7.

    van Opijnen, T., Bodi, K. L. & Camilli, A. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat. Methods 6, 767–772 (2009).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  8. 8.

    Wang, H. H. et al. Genome-scale promoter engineering by coselection MAGE. Nat. Methods 9, 591–593 (2012).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  9. 9.

    Sharan, S. K., Thomason, L. C., Kuznetsov, S. G. & Court, D. L. Recombineering: a homologous recombination-based method of genetic engineering. Nat. Protoc. 4, 206–223 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Zhang, Y., Buchholz, F., Muyrers, J. P. & Stewart, A. F. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–128 (1998).

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

  12. 12.

    Cotta-de-Almeida, V., Schonhoff, S., Shibata, T., Leiter, A. & Snapper, S. B. A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria. Genome Res. 13, 2190–2194 (2003).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR–Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. 15.

    Wang, K. et al. Defining synonymous codon compression schemes by genome recoding. Nature 539, 59–64 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Sukhija, K. et al. Developing an extended genomic engineering approach based on recombineering to knock-in heterologous genes to Escherichia coli genome. Mol. Biotechnol. 51, 109–118 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  17. 17.

    Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. 18.

    Vento, J. M., Crook, N. & Beisel, C. L. Barriers to genome editing with CRISPR in bacteria. J. Ind. Microbiol. Biotechnol. 46, 1327–1341 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Jiang, Y. et al. CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat. Commun. 8, 15179 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Wannier, T. M. et al. Improved bacterial recombineering by parallelized protein discovery. Proc. Natl Acad. Sci. USA 117, 13689–13698 (2020).

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Corts, A. D., Thomason, L. C., Gill, R. T. & Gralnick, J. A. A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides. Sci. Rep. 9, 39 (2019).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  23. 23.

    Peters, J. M. et al. Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi. Nat. Microbiol. 4, 244–250 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24.

    St-Pierre, F. et al. One-step cloning and chromosomal integration of DNA. ACS Synth. Biol. 2, 537–541 (2013).

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    Tellier, M., Bouuaert, C. C. & Chalmers, R. Mariner and the ITm superfamily of transposons. Microbiol. Spectr. 3, MDNA3–0033–2014 (2015).

  26. 26.

    van Opijnen, T. & Camilli, A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat. Rev. Microbiol. 11, 435–442 (2013).

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Haniford, D. B. & Ellis, M. J. Transposons Tn10 and Tn5. Microbiol. Spectr. 3, MDNA3–0002–2014 (2015).

  28. 28.

    Goodall, E. C. A. et al. The essential genome of Escherichia coli K-12. Mbio 9, e02096-17 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  29. 29.

    Chen, S. P. & Wang, H. H. An engineered Cas-transposon system for programmable and site-directed DNA transpositions. CRISPR J. 2, 376–394 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Bhatt, S. & Chalmers, R. Targeted DNA transposition in vitro using a dCas9-transposase fusion protein. Nucleic Acids Res. 6, 7–10 (2019).

    Google Scholar 

  31. 31.

    Enyeart, P. J., Mohr, G., Ellington, A. D. & Lambowitz, A. M. Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis. Mob. DNA 5, 2 (2014).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  32. 32.

    Esvelt, K. M. & Wang, H. H. Genome-scale engineering for systems and synthetic biology. Mol. Syst. Biol. 9, 641 (2013).

    PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Perutka, J., Wang, W., Goerlitz, D. & Lambowitz, A. M. Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes. J. Mol. Biol. 336, 421–439 (2004).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Karberg, M. et al. Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat. Biotechnol. 19, 1162–1167 (2001).

    CAS  PubMed  Article  Google Scholar 

  35. 35.

    Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S. & Sternberg, S. H. Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature 571, 219–225 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  36. 36.

    Peters, J. E., Makarova, K. S., Shmakov, S. & Koonin, E. V. Recruitment of CRISPR–Cas systems by Tn7-like transposons. Proc. Natl Acad. Sci. USA 114, E7358–E7366 (2017).

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Halpin-Healy, T. S., Klompe, S. E., Sternberg, S. H. & Fernández, I. S. Structural basis of DNA targeting by a transposon-encoded CRISPR–Cas system. Nature 577, 271–274 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  38. 38.

    Faure, G. et al. CRISPR–Cas in mobile genetic elements: counter-defence and beyond. Nat. Rev. Microbiol. 17, 513–525 (2019).

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Peters, J. E. Targeted transposition with Tn7 elements: safe sites, mobile plasmids, CRISPR/Cas and beyond. Mol. Microbiol. 112, 1635–1644 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  40. 40.

    Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science 365, 48–53 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Chavez, M. & Qi, L. S. Site-programmable transposition: shifting the paradigm for CRISPR–Cas systems. Mol. Cell. 75, 206–208 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Hou, Z. & Zhang, Y. Inserting DNA with CRISPR. Science 365, 25–26 (2019).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  43. 43.

    Ronda, C., Chen, S. P., Cabral, V., Yaung, S. J. & Wang, H. H. Metagenomic engineering of the mammalian gut microbiome in situ. Nat Meth 16, 167–170 (2019).

    CAS  Article  Google Scholar 

  44. 44.

    Stellwagen, A. E. & Craig, N. L. Avoiding self: two Tn7-encoded proteins mediate target immunity in Tn7 transposition. EMBO J. 16, 6823–6834 (1997).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45.

    Greene, E. C. & Mizuuchi, K. Target immunity during Mu DNA transposition. Transpososome assembly and DNA looping enhance MuA-mediated disassembly of the MuB target complex. Mol. Cell 10, 1367–1378 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  46. 46.

    Hagemann, A. T. & Craig, N. L. Tn7 transposition creates a hotspot for homologous recombination at the transposon donor site. Genetics 133, 9–16 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Lin, M. T. et al. In Methods in Enzymology Isotope Labeling of Biomolecules—Labeling Methods Vol. 565 (Ed. Kelman, Z.) 45–66 (Academic Press, 2015).

  48. 48.

    Hickman, A. B. & Dyda, F. DNA transposition at work. Chem. Rev. 116, 12758–12784 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  49. 49.

    Abbas, A. F., Al-Saadi, A. G. M. & Alkhudhairy, M. K. Biofilm formation and virulence determinants of Klebsiella oxytoca clinical isolates from patients with colorectal cancer. J. Gastrointest. Cancer 51, 855–860 (2019).

  50. 50.

    Kim, D.-K. et al. Metabolic engineering of a novel Klebsiella oxytoca strain for enhanced 2,3-butanediol production. J. Biosci. Bioeng. 116, 186–192 (2013).

    CAS  PubMed  Article  Google Scholar 

  51. 51.

    Loeschcke, A. & Thies, S. Pseudomonas putida—a versatile host for the production of natural products. Appl. Microbiol. Biotechnol. 99, 6197–6214 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Nikel, P. I. & de Lorenzo, V. Pseudomonas putida as a functional chassis for industrial biocatalysis: from native biochemistry to trans-metabolism. Metab. Eng. 50, 142–155 (2018).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  53. 53.

    Sun, J. et al. Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system. Microb. Cell Fact. 17, 41 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  54. 54.

    Wirth, N. T., Kozaeva, E. & Nikel, P. I. Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR–Cas9 counterselection. Microb. Biotechnol. 13, 233–249 (2020).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  55. 55.

    Tsai, S. Q. & Joung, J. K. Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases. Nat. Rev. Genet. 17, 300–312 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. 56.

    Zhang, Y. et al. Multicopy chromosomal integration using CRISPR-associated transposases. ACS Synth. Biol. 9, 1998–2008 (2020).

    CAS  PubMed  Article  Google Scholar 

  57. 57.

    Yu, B. J. & Kim, C. Minimization of the Escherichia coli genome using the Tn5-targeted Cre/loxP excision system. Nat. Biotechnol. 20, 1018–1023 (2008).

    Article  CAS  Google Scholar 

  58. 58.

    Adiego-Pérez, B. et al. Multiplex genome editing of microorganisms using CRISPR–Cas. FEMS Microbiol. Lett. 366, fnz086 (2019).

  59. 59.

    Horlbeck, M. A. et al. Mapping the genetic landscape of human cells. Cell 174, 953–967(2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  60. 60.

    Bassalo, M. C. et al. Rapid and efficient one-step metabolic pathway integration in E. coli. ACS Synth. Biol. 5, 561–568 (2016).

    CAS  PubMed  Article  Google Scholar 

  61. 61.

    Rubin, B. E. et al. Targeted genome editing of bacteria within microbial communities. Preprint at bioRxiv https://doi.org/10.1101/2020.07.17.209189 (2020).

  62. 62.

    Valderrama, J. A., Kulkarni, S. S., Nizet, V. & Bier, E. A bacterial gene-drive system efficiently edits and inactivates a high copy number antibiotic resistance locus. Nat. Commun. 10, 5726–5728 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  63. 63.

    Duque, E. et al. Identification and elucidation of in vivo function of two alanine racemases from Pseudomonas putida KT2440. Environ. Microbiol. Rep. 9, 581–588 (2017).

    CAS  PubMed  Article  Google Scholar 

  64. 64.

    Aparicio, T., de Lorenzo, V. & Martínez-García, E. CRISPR/Cas9-enhanced ssDNA recombineering for Pseudomonas putida. Microb. Biotechnol. 12, 1076–1089 (2019).

  65. 65.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  66. 66.

    Rice, P. A., Craig, N. L. & Dyda, F. Comment on ‘RNA-guided DNA insertion with CRISPR-associated transposases’. Science 368, eabb2022 (2020).

    CAS  PubMed  Article  Google Scholar 

  67. 67.

    Strecker, J., Ladha, A., Makarova, K. S., Koonin, E. V. & Zhang, F. Response to comment on ‘RNA-guided DNA insertion with CRISPR-associated transposases’. Science 368, eabb2920 (2020).

  68. 68.

    Kozich, J. J., Westcott, S. L., Baxter, N. T., Highlander, S. K. & Schloss, P. D. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl. Environ. Microbiol. 79, 5112–5120 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  69. 69.

    Callahan, B. J. et al. DADA2: high-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  70. 70.

    McMurdie, P. J. & Holmes, S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8, e61217 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

Download references

Acknowledgements

We thank N. Jaber for laboratory support, J. Bondy-Denomy for discussions, L.F. Landweber for qPCR instrument access, J. Mohabir for assistance with NGS read alignment, the JP Sulzberger Columbia Genome Center for NGS support and M.L. Smith, I. Oussenko and the Genomics Technology Laboratory at the Icahn School of Medicine at Mount Sinai for SMRT sequencing. H.H.W. acknowledges funding support for this work from the National Science Foundation (MCB-1453219), the National Institutes of Health (1U01GM110714 and 1R01AI132403), the Office of Naval Research (N00014-17-1-2353) and the Burroughs Wellcome Fund (PATH1016691). C.R. is supported by a Junior Fellows Scholarship from the Simons Society of Fellows. S.H.S. acknowledges a generous startup package from the Columbia University Irving Medical Center Dean’s Office and the Vagelos Precision Medicine Fund.

Author information

Affiliations

Authors

Contributions

P.L.H.V. and S.H.S. conceived of and designed the project, with input from C.R. and H.H.W. P.L.H.V. performed experiments and analyzed data for most E. coli experiments. C.R. performed experiments and analyzed data in K. oxytoca, P. putida and complex bacterial communities, with input from H.H.W. S.E.K. performed target immunity, ShoINT and random fragmentation NGS experiments. E.E.C. helped with cloning and transposition experiments. C.A. assisted with computational analyses of NGS data and the guide RNA design algorithm. P.L.H.V., S.H.S. and all other authors discussed the data and wrote the manuscript.

Corresponding author

Correspondence to Samuel H. Sternberg.

Ethics declarations

Competing interests

P.L.H.V., S.E.K. and S.H.S. are inventors on patents and patent applications related to CRISPR–Cas systems and uses thereof. H.H.W. is a scientific advisor to SNIPR Biome. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences and an equity holder in Dahlia Biosciences and Caribou Biosciences.

Additional information

Peer review information Nature Biotechnology thanks Joseph Bondy-Denomy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Supplementary information

Supplementary Information

Supplementary Figs. 1–15.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–7.

Source data

Source Data Fig. 3

Unprocessed gels

Source Data Fig. 4

Unprocessed gels

Source Data Fig. 5

Unprocessed gels

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Vo, P.L.H., Ronda, C., Klompe, S.E. et al. CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-00745-y

Download citation

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing