The ability to record transcriptional events within a cell over time would help to elucidate how molecular events give rise to complex cellular behaviours and states. However, current molecular recording technologies capture only a small set of defined stimuli.

Researchers at ETH Zurich developed a method they called Record-seq for capturing gene-expression profiles. They genetically engineered the bacterium Escherichia coli to contain the RNA-acquisition proteins from F. saccharivorans. They then verified that these proteins could incorporate spacers into the genetic information of the E. coli cell, and that RNA rather than DNA sequences determined the corresponding spacer DNA.

In Record-seq, the RNA-acquisition proteins are expressed during the recording of the gene-expression profile. At the end of this period, a sample of the cell population is taken. Newly expanded CRISPR arrays are isolated and sequenced, and the spacers are matched to the corresponding genomic sequences.

The next steps were to prove that the method could faithfully create a record of gene expression and to determine what could be discerned about the cellular environment during the recording period. The researchers found that Record-seq could record hundreds to thousands of different RNA transcripts present in the cell at any time. Although there was a strong bias towards capture of highly abundant transcripts, the transcript abundance of particular RNAs, as assessed by RNA sequencing, generally correlated with the frequency with which a corresponding spacer sequence was acquired in the sample. Furthermore, the collection of spacers could form a particular pattern depending on the growth conditions in which the cells were cultivated, allowing the authors to use such a spacer ‘fingerprint’ as a way to discern the conditions that the cells had experienced.

Transcriptional recording by CRISPR spacer acquisition from RNA

a, Expression of RT–Cas1–Cas2 leads to the acquisition of intracellular RNAs, providing a molecular memory of transcriptional events stored within DNA. b, Comparison of RNA-seq and Record–seq. RNA-seq captures the transcriptome of a population of cells at a single point in time, providing a transient snapshot of cellular events. By contrast, Record–seq permanently stores information about prior transcriptional events in a CRISPR array, providing a molecular record that can be used to reconstruct transcriptional events that occurred over time.

In the future, CRISPR spacer acquisition-mediated recording of RNA followed by deep sequencing (Record–seq) could be used to reconstruct transcriptional histories that describe complex cell behaviours or pathological states.

Availability – Record-seq code Scripts, example data, and workflow download (ZIP, 10 MB)

Source – ETH Zurich