Recording Gene Expression Order in DNA by CRISPR Addition of Retron Barcodes

Overview

Journal Nature

Specialty Science

Date 2022 Jul 27

PMID 35896746

Authors

Santi Bhattarai-Kline

Sierra K Lear

Chloe B Fishman

Santiago C Lopez

Elana R Lockshin

Max G Schubert

Jeff Nivala

George M Church

Seth L Shipman

Affiliations

Soon will be listed here.

Abstract

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, that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system. 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.

Citing Articles

Advances and applications in single-cell and spatial genomics.

Wang J, Ye F, Chai H, Jiang Y, Wang T, Ran X Sci China Life Sci. 2025; .

PMID: 39792333 DOI: 10.1007/s11427-024-2770-x.

Induced cell phenotype activity recording of DNA-tagged ligands.

Sander P, Gillen Miller J, Lairson L RSC Chem Biol. 2025; 6(2):273-280.

PMID: 39776788 PMC: 11701724. DOI: 10.1039/d4cb00137k.

High throughput variant libraries and machine learning yield design rules for retron gene editors.

Crawford K, Khan A, Lopez S, Goodarzi H, Shipman S Nucleic Acids Res. 2024; 53(2.

PMID: 39658047 PMC: 11754653. DOI: 10.1093/nar/gkae1199.

The lives of cells, recorded.

Askary A, Chen W, Choi J, Du L, Elowitz M, Gagnon J Nat Rev Genet. 2024; 26(3):203-222.

PMID: 39587306 DOI: 10.1038/s41576-024-00788-w.

Barcoding Notch signaling in the developing brain.

Siniscalco A, Perera R, Greenslade J, Veeravenkatasubramanian H, Masters A, Doll H Development. 2024; 151(24).

PMID: 39575683 PMC: 11701514. DOI: 10.1242/dev.203102.

References

Roquet N, Soleimany A, Ferris A, Aaronson S, Lu T . Synthetic recombinase-based state machines in living cells. Science. 2016; 353(6297):aad8559. DOI: 10.1126/science.aad8559. View

Bonnet J, Subsoontorn P, Endy D . Rewritable digital data storage in live cells via engineered control of recombination directionality. Proc Natl Acad Sci U S A. 2012; 109(23):8884-9. PMC: 3384180. DOI: 10.1073/pnas.1202344109. View

Ceze L, Nivala J, Strauss K . Molecular digital data storage using DNA. Nat Rev Genet. 2019; 20(8):456-466. DOI: 10.1038/s41576-019-0125-3. View

Meyer A, Segall-Shapiro T, Glassey E, Zhang J, Voigt C . Escherichia coli "Marionette" strains with 12 highly optimized small-molecule sensors. Nat Chem Biol. 2018; 15(2):196-204. DOI: 10.1038/s41589-018-0168-3. View

Lampson B, Sun J, Hsu M, Inouye S, Inouye M . Reverse transcriptase in a clinical strain of Escherichia coli: production of branched RNA-linked msDNA. Science. 1989; 243(4894 Pt 1):1033-8. DOI: 10.1126/science.2466332. View

Rogers J, Guzman C, Taylor N, Raman S, Anderson K, Church G . Synthetic biosensors for precise gene control and real-time monitoring of metabolites. Nucleic Acids Res. 2015; 43(15):7648-60. PMC: 4551912. DOI: 10.1093/nar/gkv616. View

Church G, Gao Y, Kosuri S . Next-generation digital information storage in DNA. Science. 2012; 337(6102):1628. DOI: 10.1126/science.1226355. View

Millman A, Bernheim A, Stokar-Avihail A, Fedorenko T, Voichek M, Leavitt A . Bacterial Retrons Function In Anti-Phage Defense. Cell. 2020; 183(6):1551-1561.e12. DOI: 10.1016/j.cell.2020.09.065. View

Datsenko K, Wanner B . One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000; 97(12):6640-5. PMC: 18686. DOI: 10.1073/pnas.120163297. View

10.

Lim D . Structure and biosynthesis of unbranched multicopy single-stranded DNA by reverse transcriptase in a clinical Escherichia coli isolate. Mol Microbiol. 1992; 6(23):3531-42. DOI: 10.1111/j.1365-2958.1992.tb01788.x. View

11.

Schubert M, Goodman D, Wannier T, Kaur D, Farzadfard F, Lu T . High-throughput functional variant screens via in vivo production of single-stranded DNA. Proc Natl Acad Sci U S A. 2021; 118(18). PMC: 8106316. DOI: 10.1073/pnas.2018181118. View

12.

Yim S, McBee R, Song A, Huang Y, Sheth R, Wang H . Robust direct digital-to-biological data storage in living cells. Nat Chem Biol. 2021; 17(3):246-253. PMC: 7904632. DOI: 10.1038/s41589-020-00711-4. View

13.

Wagner D, Klein A . Lineage tracing meets single-cell omics: opportunities and challenges. Nat Rev Genet. 2020; 21(7):410-427. PMC: 7307462. DOI: 10.1038/s41576-020-0223-2. View

14.

Lopez S, Crawford K, Lear S, Bhattarai-Kline S, Shipman S . Precise genome editing across kingdoms of life using retron-derived DNA. Nat Chem Biol. 2021; 18(2):199-206. PMC: 8810715. DOI: 10.1038/s41589-021-00927-y. View

15.

Yosef I, Goren M, Qimron U . Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 2012; 40(12):5569-76. PMC: 3384332. DOI: 10.1093/nar/gks216. View

16.

Hayflick L, Moorhead P . The serial cultivation of human diploid cell strains. Exp Cell Res. 1961; 25:585-621. DOI: 10.1016/0014-4827(61)90192-6. View

17.

Kim S, Loeff L, Colombo S, Jergic S, Brouns S, Joo C . Selective loading and processing of prespacers for precise CRISPR adaptation. Nature. 2020; 579(7797):141-145. DOI: 10.1038/s41586-020-2018-1. View

18.

Street K, Risso D, Fletcher R, Das D, Ngai J, Yosef N . Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics. BMC Genomics. 2018; 19(1):477. PMC: 6007078. DOI: 10.1186/s12864-018-4772-0. View

19.

Ramachandran A, Summerville L, Learn B, DeBell L, Bailey S . Processing and integration of functionally oriented prespacers in the CRISPR system depends on bacterial host exonucleases. J Biol Chem. 2020; 295(11):3403-3414. PMC: 7076208. DOI: 10.1074/jbc.RA119.012196. View

20.

Simon A, Ellington A, Finkelstein I . Retrons and their applications in genome engineering. Nucleic Acids Res. 2019; 47(21):11007-11019. PMC: 6868368. DOI: 10.1093/nar/gkz865. View