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New molecular device ‘Retro-Cascorder’ uses CRISPR to track and record gene expression over time

Written by Miyako Rogers, Science Writer

In a recent paper published in Nature, researchers developed a molecular device called “Retro-Cascorder”, which makes sequential recordings of transcriptional events. Using CRISPR-Cas integrases to incorporate retron barcodes into a cell’s genome, Retro-Cascorder logs receipts of gene expression. This will allow researchers to uncover the history of gene expression in a living cell, unlocking insights into many different biological processes.

Recording gene-expression events over time

All biological processes depend on the differential expression of genes over time, but previous methods to make time-ordered recordings of these changes are limited, relying on inferential statistics and using assumptions based on prior knowledge. Molecular recorders do not rely on inference, using CRISPR-Cas integrases or nucleases to continuously record gene-expression changes. They then permanently store them in a physical record, using DNA as their storage medium.

However, existing molecular recorders can only record one event at a time. Retro-Cascorder overcomes this limitation by adding retrons to the gene of interest: These are engineered RNA barcodes based on prokaryotic (bacterial) elements that produce specific DNA sequences when activated.

Developing Retro-Cascorder

E.coli was used to develop Retro-Cascorder. Retrons were engineered to produce a specific tag sequence and then placed under the control of promoter sequences for specific genes of interest. When the promoter is activated, the tag sequence is transcribed to RNA. This RNA sequence is then reverse transcribed by the retron reverse-transcriptase contained within Retro-Cascorder, generating a DNA receipt. CRISPR integrases then integrate the DNA receipt into a CRISR array, thus creating a record of transcription. CRISPR arrays contain spacer sequences and if another promoter is then activated, this process repeats, placing the new DNA receipt after the first spacer.

Figure 1 | Cas1-Cas2 integrates retron RT-DNA. A: Illustration of retroelement-based transcriptional recording into CRISPR arrays. B: Illustration of biological components contained in the retron-based recorder. (Source: Nature)

To test Retro-Cascorder, researchers tagged specific genes in E.coli, which are known to activate in the presence of specific chemicals. Over 48 hours, transcription events were recorded and the order of DNA receipts in the CRISPR array corresponded to the order in which the chemicals were applied. Thus, Retro-Cascorder records the temporal history of specific gene transcription events.

Future uses and applications

At the moment, Retro-Cascorder has only been used in bacteria and only shows the order in which genes are transcribed, not the time between each event. However, this information can still be used to create living biosensors, which can sample and record changes in a given environment. These sensors could be used in wastewater facilities to detect for contaminants, in ponds and other outside environments to test for pollutants and even in the human gut to detect pathogens. In the future, as this technology continues to be developed, Retro-Cascorder could also be used to record gene expression patterns during complex cell events, such as in immune cells during an inflammatory response or in cancer cells to unravel the process of tumour formation.

Image Credit: Canva

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