Recording gene expression order in DNA by CRISPR addition of retron barcodes.

Publisher: Nature Publishing Group Country of Publication: England NLM ID: 0410462 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1476-4687 (Electronic) Linking ISSN: 00280836 NLM ISO Abbreviation: Nature Subsets: MEDLINE

Publication: Basingstoke : Nature Publishing Group
Original Publication: London, Macmillan Journals ltd.

CRISPR-Cas Systems*/genetics ; DNA*/biosynthesis ; DNA*/genetics ; Gene Editing*/methods ; Gene Expression* ; Information Storage and Retrieval*/methods ; RNA*/genetics ; Reverse Transcription*; Genome/genetics ; Integrases/metabolism ; Prokaryotic Cells/metabolism ; Time Factors

Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this through engineered RNA barcodes, based on prokaryotic retrons ¹ , that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system ² . The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record through simple, logical rules rather than relying on pretrained classifiers or post hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.
(© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Comment in: Nat Methods. 2022 Sep;19(9):1031. (PMID: 36068315)

Simon, A. J., Ellington, A. D. & Finkelstein, I. J. Retrons and their applications in genome engineering. Nucleic Acids Res. 47, 11007–11019 (2019). (PMID: 31598685686836810.1093/nar/gkz865)
Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007). (PMID: 1737980810.1126/science.1138140)
Church, G. M., Gao, Y. & Kosuri, S. Next-generation digital information storage in DNA. Science 337, 1628–1628 (2012). (PMID: 2290351910.1126/science.1226355)
Shipman, S. L., Nivala, J., Macklis, J. D. & Church, G. M. CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria. Nature 547, 345–349 (2017). (PMID: 28700573584279110.1038/nature23017)
Yim, S. S. et al. Robust direct digital-to-biological data storage in living cells. Nat. Chem. Biol. 17, 246–253 (2021). (PMID: 33432236790463210.1038/s41589-020-00711-4)
Ceze, L., Nivala, J. & Strauss, K. Molecular digital data storage using DNA. Nat. Rev. Genet. 20, 456–466 (2019). (PMID: 3106868210.1038/s41576-019-0125-3)
Roquet, N., Soleimany, A. P., Ferris, A. C., Aaronson, S. & Lu, T. K. Synthetic recombinase-based state machines in living cells. Science 353, aad8559 (2016). (PMID: 2746367810.1126/science.aad8559)
Sheth, R. U., Yim, S. S., Wu, F. L. & Wang, H. H. Multiplex recording of cellular events over time on CRISPR biological tape. Science 358, 1457–1461 (2017). (PMID: 29170279786911110.1126/science.aao0958)
Schmidt, F., Cherepkova, M. Y. & Platt, R. J. Transcriptional recording by CRISPR spacer acquisition from RNA. Nature 562, 380–385 (2018). (PMID: 3028313510.1038/s41586-018-0569-1)
Wagner, D. E. & Klein, A. M. Lineage tracing meets single-cell omics: opportunities and challenges. Nat. Rev. Genet. 21, 410–427 (2020). (PMID: 32235876730746210.1038/s41576-020-0223-2)
Street, K. et al. Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics. BMC Genomics 19, 477 (2018). (PMID: 29914354600707810.1186/s12864-018-4772-0)
Perli, S. D., Cui, C. H. & Lu, T. K. Continuous genetic recording with self-targeting CRISPR–Cas in human cells. Science 353, aag0511 (2016). (PMID: 2754000610.1126/science.aag0511)
Park, J. et al. Recording of elapsed time and temporal information about biological events using Cas9. Cell 184, 1047–1063 (2021). (PMID: 3353978010.1016/j.cell.2021.01.014)
Shipman, S. L., Nivala, J., Macklis, J. D. & Church, G. M. Molecular recordings by directed CRISPR spacer acquisition. Science 353, aaf1175 (2016). (PMID: 27284167499489310.1126/science.aaf1175)
Simon, A. J., Morrow, B. R. & Ellington, A. D. Retroelement-based genome editing and evolution. ACS Synth. Biol. 7, 2600–2611 (2018). (PMID: 3025662110.1021/acssynbio.8b00273)
Sharon, E. et al. Functional genetic variants revealed by massively parallel precise genome editing. Cell 175, 544–557 (2018). (PMID: 30245013656382710.1016/j.cell.2018.08.057)
Schubert, M. G. et al. High-throughput functional variant screens via in vivo production of single-stranded DNA. Proc. Natl Acad. Sci. USA 118, e2018181118 (2021). (PMID: 33906944810631610.1073/pnas.2018181118)
Lopez, S. C., Crawford, K. D., Lear, S. K., Bhattarai-Kline, S. & Shipman, S. L. Precise genome editing across kingdoms of life using retron-derived DNA. Nat. Chem. Biol. 18, 199–206 (2022). (PMID: 3494983810.1038/s41589-021-00927-y)
Farzadfard, F. & Lu, T. K. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations. Science 346, 1256272 (2014). (PMID: 25395541426647510.1126/science.1256272)
Yosef, I., Goren, M. G. & Qimron, U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 40, 5569–5576 (2012). (PMID: 22402487338433210.1093/nar/gks216)
Nuñez, J. K. et al. Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity. Nat. Struct. Mol. Biol. 21, 528–534 (2014). (PMID: 24793649407594210.1038/nsmb.2820)
Wang, J. et al. Structural and mechanistic basis of PAM-dependent spacer acquisition in CRISPR–Cas systems. Cell 163, 840–853 (2015). (PMID: 2647818010.1016/j.cell.2015.10.008)
Millman, A. et al. Bacterial retrons function in anti-phage defense. Cell 183, 1551–1561 (2020). (PMID: 3315703910.1016/j.cell.2020.09.065)
Bobonis, J. et al. Bacterial retrons encode tripartite toxin/antitoxin systems. Preprint at bioRxiv https://doi.org/10.1101/2020.06.22.160168 (2020).
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). (PMID: 246633210.1126/science.2466332)
Silas, S. et al. Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase–Cas1 fusion protein. Science 351, aad4234 (2016). (PMID: 26917774489865610.1126/science.aad4234)
Bonnet, J., Subsoontorn, P. & Endy, D. Rewritable digital data storage in live cells via engineered control of recombination directionality. Proc. Natl Acad. Sci. USA 109, 8884–8889 (2012). (PMID: 22615351338418010.1073/pnas.1202344109)
Kim, S. et al. Selective loading and processing of prespacers for precise CRISPR adaptation. Nature 579, 141–145 (2020). (PMID: 3207626210.1038/s41586-020-2018-1)
Ramachandran, A., Summerville, L., Learn, B. A., DeBell, L. & Bailey, S. Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases. J. Biol. Chem. 295, 3403–3414 (2020). (PMID: 3191441810.1074/jbc.RA119.012196)
Chapman, K. B. & Boeke, J. D. Isolation and characterization of the gene encoding yeast debranching enzyme. Cell 65, 483–492 (1991). (PMID: 185032310.1016/0092-8674(91)90466-C)
Lim, D. Structure and biosynthesis of unbranched multicopy single-stranded DNA by reverse transcriptase in a clinical Eschehchia coli isolate. Mol. Microbiol. 6, 3531–3542 (1992). (PMID: 128219110.1111/j.1365-2958.1992.tb01788.x)
Jung, H., Liang, J., Jung, Y. & Lim, D. Characterization of cell death in Escherichia coli mediated by XseA, a large subunit of exonuclease VII. J. Microbiol. 53, 820–828 (2015). (PMID: 2662635210.1007/s12275-015-5304-0)
Han, E. S. et al. RecJ exonuclease: substrates, products and interaction with SSB. Nucleic Acids Res. 34, 1084–1091 (2006). (PMID: 16488881137369210.1093/nar/gkj503)
Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J. & Voigt, C. A. Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nat. Chem. Biol. 15, 196–204 (2019). (PMID: 3047845810.1038/s41589-018-0168-3)
Grubbs, F. E. Procedures for detecting outlying observations in samples. Technometrics 11, 1–21 (1969). (PMID: 10.1080/00401706.1969.10490657)
Stefansky, W. Rejecting outliers in factorial designs. Technometrics 14, 469–479 (1972). (PMID: 10.1080/00401706.1972.10488930)
Hayflick, L. & Moorhead, P. S. The serial cultivation of human diploid cell strains. Exp. Cell. Res. 25, 585–621 (1961). (PMID: 1390565810.1016/0014-4827(61)90192-6)
Yang, L. et al. Permanent genetic memory with >1-byte capacity. Nat. Methods 11, 1261–1266 (2014). (PMID: 25344638424532310.1038/nmeth.3147)
Yehl, K. & Lu, T. Scaling computation and memory in living cells. Curr. Opin. Biomed. Eng. 4, 143–151 (2017). (PMID: 29915814600371810.1016/j.cobme.2017.10.003)
Mosberg, J. A., Gregg, C. J., Lajoie, M. J., Wang, H. H. & Church, G. M. Improving lambda Red genome engineering in Escherichia coli via rational removal of endogenous nucleases. PLoS ONE 7, e44638 (2012). (PMID: 22957093343416510.1371/journal.pone.0044638)
Moore, S. D. In Strain Engineering: Methods and Protocols (ed. Williams, J. A.) 155–169 (Humana Press, 2011).
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). (PMID: 108290791868610.1073/pnas.120163297)
Rogers, J. K. et al. Synthetic biosensors for precise gene control and real-time monitoring of metabolites. Nucleic Acids Res. 43, 7648–7660 (2015). (PMID: 26152303455191210.1093/nar/gkv616)

DP2 GM140917 United States GM NIGMS NIH HHS

63231-63-0 (RNA)
9007-49-2 (DNA)
EC 2.7.7.- (Integrases)

Date Created: 20220727 Date Completed: 20220808 Latest Revision: 20230413

20240104

PMC9357182

10.1038/s41586-022-04994-6

35896746