Temporally resolved transcriptional recording in E. coli DNA using a Retro-Cascorder.

Autor: Lear SK; Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA.; UCSF-UCB Graduate Program in Bioengineering, University of California, Berkeley, CA, USA., Lopez SC; Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA.; UCSF-UCB Graduate Program in Bioengineering, University of California, Berkeley, CA, USA., González-Delgado A; Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA., Bhattarai-Kline S; Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA.; UCLA-Caltech Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA., Shipman SL; Gladstone Institute of Data Science and Biotechnology, San Francisco, CA, USA. seth.shipman@gladstone.ucsf.edu.; Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA. seth.shipman@gladstone.ucsf.edu.; Chan Zuckerberg Biohub, San Francisco, CA, USA. seth.shipman@gladstone.ucsf.edu.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2023 Jun; Vol. 18 (6), pp. 1866-1892. Date of Electronic Publication: 2023 Apr 14.
DOI: 10.1038/s41596-023-00819-6
Abstrakt: Biological signals occur over time in living cells. Yet most current approaches to interrogate biology, particularly gene expression, use destructive techniques that quantify signals only at a single point in time. A recent technological advance, termed the Retro-Cascorder, overcomes this limitation by molecularly logging a record of gene expression events in a temporally organized genomic ledger. The Retro-Cascorder works by converting a transcriptional event into a DNA barcode using a retron reverse transcriptase and then storing that event in a unidirectionally expanding clustered regularly interspaced short palindromic repeats (CRISPR) array via acquisition by CRISPR-Cas integrases. This CRISPR array-based ledger of gene expression can be retrieved at a later point in time by sequencing. Here we describe an implementation of the Retro-Cascorder in which the relative timing of transcriptional events from multiple promoters of interest is recorded chronologically in Escherichia coli populations over multiple days. We detail the molecular components required for this technology, provide a step-by-step guide to generate the recording and retrieve the data by Illumina sequencing, and give instructions for how to use custom software to infer the relative transcriptional timing from the sequencing data. The example recording is generated in 2 d, preparation of sequencing libraries and sequencing can be accomplished in 2-3 d, and analysis of data takes up to several hours. This protocol can be implemented by someone familiar with basic bacterial culture, molecular biology and bioinformatics. Analysis can be minimally run on a personal computer.
(© 2023. Springer Nature Limited.)
Databáze: MEDLINE
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