Temporally resolved transcriptional recordings into E. coli DNA using a Retro-Cascorder
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...
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Veröffentlicht in: | Nature protocols 2023-04, Vol.18 (6), p.1866-1892 |
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Sprache: | eng |
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Zusammenfassung: | 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 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 two days, preparation of sequencing libraries and sequencing can be accomplished in two to three days, 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.
This protocol describes Retro-Cascorder to make temporally resolved transcriptional recordings in
E. coli
DNA, using a retron reverse transcriptase to store transcriptional events in a unidirectionally-expanding CRISPR array via acquisition by CRISPR-Cas integrases. |
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ISSN: | 1754-2189 1750-2799 |
DOI: | 10.1038/s41596-023-00819-6 |