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Precise genome editing across kingdoms of life using retron-derived DNA

Abstract

Exogenous DNA can be a template to precisely edit a cell’s genome. However, the delivery of in vitro-produced DNA to target cells can be inefficient, and low abundance of template DNA may underlie the low rate of precise editing. One potential tool to produce template DNA inside cells is a retron, a bacterial retroelement involved in phage defense. However, little effort has been directed at optimizing retrons to produce designed sequences. Here, we identify modifications to the retron non-coding RNA (ncRNA) that result in more abundant reverse-transcribed DNA (RT-DNA). By testing architectures of the retron operon that enable efficient reverse transcription, we find that gains in DNA production are portable from prokaryotic to eukaryotic cells and result in more efficient genome editing. Finally, we show that retron RT-DNA can be used to precisely edit cultured human cells. These experiments provide a general framework to produce DNA using retrons for genome modification.

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Fig. 1: Bacterial retrons enable RT-DNA production.
Fig. 2: Modifications to retron ncRNA affect RT-DNA production.
Fig. 3: RT-DNA production in eukaryotic cells.
Fig. 4: Improvements extend to applications in genome editing.
Fig. 5: Precise editing by retrons extends to human cells.

Data availability

All data supporting the findings of this study are available within the article and its Supplementary Information. Sequencing data associated with this study are available through the NCBI BioProject database under accession number PRJNA770365. Source data are provided with this paper.

Code availability

Custom code to process or analyze data from this study is available on GitHub at https://github.com/Shipman-Lab/retron_architectures.

References

  1. Luo, D. & Saltzman, W. M. Synthetic DNA delivery systems. Nat. Biotechnol. 18, 33–37 (2000).

    Article  CAS  Google Scholar 

  2. Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

    Article  Google Scholar 

  3. Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).

    Article  CAS  Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

  5. Devkota, S. The road less traveled: strategies to enhance the frequency of homology-directed repair (HDR) for increased efficiency of CRISPR/Cas-mediated transgenesis. BMB Rep. 51, 437–443 (2018).

    Article  CAS  Google Scholar 

  6. Farzadfard, F. & Lu, T. K. Synthetic biology. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations. Science 346, 1256272 (2014).

    Article  Google Scholar 

  7. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    Article  CAS  Google Scholar 

  8. Sharon, E. et al. Functional genetic variants revealed by massively parallel precise genome editing. Cell 175, 544–557 (2018).

    Article  CAS  Google Scholar 

  9. Schubert, M. G. et al. High throughput functional variant screens via in-vivo production of single-stranded DNA. Proc. Natl Acad. Sci. 118, e2018181118 (2021).

    Article  CAS  Google Scholar 

  10. Millman, A. et al. Bacterial retrons function in anti-phage defense. Cell 183, 1551–1561 (2020).

    Article  CAS  Google Scholar 

  11. Bobonis, J. et al. Bacterial retrons encode tripartite toxin/antitoxin systems. Preprint at bioRxiv https://doi.org/10.1101/2020.06.22.160168 (2020).

  12. Bobonis, J. et al. Phage proteins block and trigger retron toxin/antitoxin systems. Preprint at bioRxiv https://doi.org/10.1101/2020.06.22.160242 (2020).

  13. Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084 (2020).

    Article  CAS  Google Scholar 

  14. Mirochnitchenko, O., Inouye, S. & Inouye, M. Production of single-stranded DNA in mammalian cells by means of a bacterial retron. J. Biol. Chem. 269, 2380–2383 (1994).

    Article  CAS  Google Scholar 

  15. Lampson, B. C., Inouye, M. & Inouye, S. Retrons, msDNA, and the bacterial genome. Cytogenet. Genome Res. 110, 491–499 (2005).

    Article  CAS  Google Scholar 

  16. Simon, A. J., Ellington, A. D. & Finkelstein, I. J. Retrons and their applications in genome engineering. Nucleic Acids Res. 47, 11007–11019 (2019).

    Article  CAS  Google Scholar 

  17. Inouye, S., Hsu, M. Y., Eagle, S. & Inouye, M. Reverse transcriptase associated with the biosynthesis of the branched RNA-linked msDNA in Myxococcus xanthus. Cell 56, 709–717 (1989).

    Article  CAS  Google Scholar 

  18. Lampson, B. C., Inouye, M. & Inouye, S. Reverse transcriptase with concomitant ribonuclease H activity in the cell-free synthesis of branched RNA-linked msDNA of Myxococcus xanthus. Cell 56, 701–707 (1989).

    Article  CAS  Google Scholar 

  19. Lampson, B. C. et al. Reverse transcriptase in a clinical strain of Escherichia coli: production of branched RNA-linked msDNA. Science 243, 1033–1038 (1989).

    Article  CAS  Google Scholar 

  20. Lim, D. & Maas, W. K. Reverse transcriptase-dependent synthesis of a covalently linked, branched DNA–RNA compound in E. coli B. Cell 56, 891–904 (1989).

    Article  CAS  Google Scholar 

  21. Miyata, S., Ohshima, A., Inouye, S. & Inouye, M. In vivo production of a stable single-stranded cDNA in Saccharomyces cerevisiae by means of a bacterial retron. Proc. Natl Acad. Sci. USA 89, 5735–5739 (1992).

    Article  CAS  Google Scholar 

  22. Chappell, S. A., Edelman, G. M. & Mauro, V. P. Ribosomal tethering and clustering as mechanisms for translation initiation. Proc. Natl Acad. Sci. USA 103, 18077–18082 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Aronshtam, A. & Marinus, M. G. Dominant negative mutator mutations in the mutL gene of Escherichia coli. Nucleic Acids Res. 24, 2498–2504 (1996).

    Article  CAS  Google Scholar 

  25. Nyerges, Á. et al. A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. Proc. Natl Acad. Sci. USA 113, 2502–2507 (2016).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Zhang, Y. et al. A gRNA–tRNA array for CRISPR–Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae. Nat. Commun. 10, 1053 (2019).

    Article  Google Scholar 

  28. Liu, J. J. et al. CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature 566, 218–223 (2019).

    Article  CAS  Google Scholar 

  29. Knapp, D. et al. Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression. Nat. Commun. 10, 1490 (2019).

    Article  Google Scholar 

  30. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  Google Scholar 

  31. Kong, X. et al. Precise genome editing without exogenous donor DNA via retron editing system in human cells. Protein Cell https://doi.org/10.1007/s13238-021-00862-7 (2021).

  32. Rogers, J. K. et al. Synthetic biosensors for precise gene control and real-time monitoring of metabolites. Nucleic Acids Res. 43, 7648–7660 (2015).

    Article  CAS  Google Scholar 

  33. 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).

    Article  CAS  Google Scholar 

  34. Gietz, R. D. & Schiestl, R. H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2, 31–34 (2007).

    Article  CAS  Google Scholar 

  35. Brachmann, C. B. et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132 (1998).

    Article  CAS  Google Scholar 

  36. Tian, S. & Das, R. Primerize-2D: automated primer design for RNA multidimensional chemical mapping. Bioinformatics 33, 1405–1406 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L. & Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR–Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by funding from the Simons Foundation Autism Research Initiative (SFARI) Bridge to Independence Award Program, the Pew Biomedical Scholars Program and a UCSF Program for Breakthrough Biomedical Research New Frontiers Research Award. S.L.S. acknowledges additional funding support from NIH/NIGMS (1DP2GM140917-01) and the L.K. Whittier Foundation. S.C.L. was supported by a Berkeley Fellowship for Graduate Study. K.D.C. was supported by an NSF Graduate Research Fellowship (2019247827) and a UCSF Discovery Fellowship. S.K.L. was supported by an NSF Graduate Research Fellowship (2034836). We thank K. Claiborn for editorial assistance.

Author information

Authors and Affiliations

Authors

Contributions

S.L.S., S.C.L. and K.D.C. conceived the study. S.C.L., K.D.C., S.K.L., S.B.-K. and S.L.S. designed the work, performed experiments, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Seth L. Shipman.

Ethics declarations

Competing interests

S.L.S., S.C.L. and K.D.C. are inventors on patent applications related to the technologies described in this work.

Additional information

Peer review information Nature Chemical Biology thanks Channabasavaiah Gurumurthy 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.

Extended data

Extended Data Fig. 1 RT-DNA sequencing prep.

a. Schematic of the sequencing prep pipeline for RT-DNA. b. Representative image of a PAGE analysis showing the addition of nucleotides to the 3’ end of a single-stranded DNA, controlled by reaction time. The experiment was repeated twice with similar results. c. Alternate analysis of the RT-DNA for the a1/a2 length library, using a TdT-based sequencing preparation. Related to Fig. 2.

Source data

Extended Data Fig. 2 RT-DNA production in eukaryotic cells.

a. Representative image of a PAGE analysis of Eco1 and Eco2 RT-DNA isolated from yeast. The ladder is shown at a different exposure to the left of the gel image. The experiment was repeated twice with similar results. b. Enrichment of the Eco1 RT-DNA/plasmid template when uninduced compared to a dead RT construct. Closed circles show each of three biological replicates, with red for the dead RT version and black for the live RT. c. Identical analysis as in b, but for Eco1 in HEK293T cells. Related to Fig. 3.

Source data

Extended Data Fig. 3 Precise genome editing rates across additional genomic loci in E. coli.

a-c. Percent of cells precisely edited, quantified by multiplexed sequencing, for the wt (black) and extended (green) recombineering constructs for three additional loci in E coli. Related to Fig. 4a–d.

Extended Data Fig. 4 Imprecise editing profile of the yeast ADE2 locus.

a. Percent of ADE2 loci with imprecise edits or sequencing errors at 24 and 48 hours. Closed circles show each of three biological replicates, with black for the wt a1/a2 length and green for the extended a1/a2 (two extended versions, v1 and v2). Induction conditions are shown below the graph for the RT and Cas9. b. Breakdown of the data in a. by type of edit/error. c. Imprecise edits and sequencing errors found in all data sets, ranked by frequency. Above the graph are the wt ADE2 locus and intended precise edit. On the Y axis are the imprecise edits and sequencing errors found. X axis represents count of each sequence in all data sets. Related to Fig. 4h.

Extended Data Fig. 5 Genome editing rates across additional genomic loci in yeast.

a-d. Percent of cells precisely edited, quantified by multiplexed sequencing, for the wt (black) and extended (green) recombineering constructs for four additional loci in S. cerevisiae at 24 and 48 hours. Cultures edited at the LYP1 E27X site were not viable beyond 24 hours. e-h. Percent of imprecise edits or sequencing errors for the loci in a-d. Related to Fig. 4e–h.

Extended Data Fig. 6 Imprecise editing rates across genomic loci in human cells.

a-f. Percent of cells imprecisely edited (indels), quantified by multiplexed sequencing, in the presence of the ncRNA/gRNA plasmid and either Cas9 alone or Cas9 and Eco1 RT (as indicated below). Individual circles represent each of three biological replicates. Related to Fig. 5.

Supplementary information

Supplementary Information

Supplementary Tables 1–4.

Reporting Summary

Supplementary Data 1

Semiprocessed data underlying Figs. 1–5. The .csv files are named according to the figures.

Supplementary Data 2

Library variant parts.

Source data

Source Data Fig. 1

Uncropped gel for Fig. 1b.

Source Data Fig. 4

Uncropped gel for Fig. 4c.

Source Data Extended Data Fig. 1

Uncropped gel for Extended Data Fig. 1b.

Source Data Extended Data Fig. 2

Uncropped gel for Extended Data Fig. 2a.

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Lopez, S.C., Crawford, K.D., Lear, S.K. et al. Precise genome editing across kingdoms of life using retron-derived DNA. Nat Chem Biol 18, 199–206 (2022). https://doi.org/10.1038/s41589-021-00927-y

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