Recording transcriptional histories using Record-seq

Abstract

It is difficult to elucidate the transcriptional history of a cell using current experimental approaches as they are destructive in nature and therefore only describe a moment in time. Overcoming these limitations, we recently established Record-seq, a technology that enables transcriptional recording by CRISPR spacer acquisition from RNA. The recorded transcriptomes are recovered by SENECA, a method that selectively amplifies expanded CRISPR arrays, followed by deep sequencing. The resulting CRISPR spacers are aligned to the host genome, thereby enabling transcript quantification and associated analyses. Here, we describe the experimental procedures of the Record-seq workflow as well as subsequent data analysis. Beginning with the experimental design, Record-seq data can be obtained and analyzed within 1-2 weeks. CRISPR spacer acquisition from RNAIn order to overcome prior limitations and enable molecular recording and DNA writing on a massive scale we developed Record-seq 17 , an approach that leverages CRISPR spacer acquisition from RNA. This was initially achieved using Cas1 and Cas2 orthologs derived from the type III CRISPR system of the human commensal bacterium Fusicatenibacter saccharivorans. The most important hallmark of the Cas1-Cas2 complex in this species is the naturally occurring fusion of Cas1 to a reverse transcriptase (RT-Cas1). Spacer acquisition from RNA presumably occurs through the binding of RT-Cas1-Cas2 to intracellular singlestranded RNA molecules and subsequent ligation of this RNA into the CRISPR array followed by reverse transcription 14 .Due to the low spacer acquisition frequencies in RNA-adapting CRISPR systems, compared to their DNA adapting counterparts, RNA spacer acquisition had not been used for DNA writing applications before the development of Record-seq. Leveraging overexpression of FsRT-Cas1-Cas2 in an E. coli host and using a newly developed protocol for the selective PCR amplification of expanded CRISPR arrays, Record-seq overcomes this low efficiency limitation and thus enables the direct recording of intracellular RNAs. Utilizing this technology, we demonstrated the recording of stimuli that transiently altered the cellular transcriptome but remained refractory to detection by traditional RNA-seq. Development of the Record-seq protocolHere we describe a detailed protocol for recording transcriptional events into plasmid-borne CRISPR arrays, named Record-seq. During bacterial growth, RNA-derived spacers are integrated into plasmid-encoded CRISPR arrays and reverse transcribed by the RT domain fused to Cas1, thereby achieving permanent storage of transcriptional events in the form of plasmid DNA (Figure 1a). The Record-seq protocol entails extracting this plasmid DNA and retrieving the spacer sequences by means of selective amplification, size selection, and deep sequencing (Figure 1b). Since only a small fraction of CRISPR arrays acquire new spacers during the course of the experiment, Record-seq incorporates a newly developed procedure to selectively amplify newly acqu...

Noninvasive assessment of gut function using transcriptional recording sentinel cells

“…The functional characteristics of Fs RT-Cas1–Cas2 (fig. S1, D to H) were consistent with our previous in vitro experiments ( 17 , 18 ).…”

The Intestinal Epithelium – Fluid Fate and Rigid Structure From Crypt Bottom to Villus Tip

“…Recently, Platt and his team made use of the system’s capacity to acquire RNA and integrate it into a CRISPR array in a sequential manner. This approach makes it possible to sample a cell’s RNA pool upon activation and store the information genetically ( Tanna et al, 2020 ). Current applications of the method are still limited to bacteria and the obtained information is very sparse, but this promising technology could pave (upon further development) the way to internal recording of the transcriptional states of mammalian cells ( Schmidt et al, 2018 ).…”

Novel Identification of Bacterial Epigenetic Regulations Would Benefit From a Better Exploitation of Methylomic Data

“…Importantly, two tools were recently designed to study complete gene expression at the subpopulation level: (i) Recordseq (Schmidt et al, 2018) can report a change in gene expression during a bottleneck situation where the amount of events is too low for a classic global transcriptomic analysis (Schmidt et al, 2018;Tanna et al, 2020); (ii) PETRIseq (Blattman et al, 2020) allows to detect subpopulations with a different transcriptomic profile where less mRNA is needed, compared to classic transcriptional studies. Combining the SMALR and one of those transcriptomic tools should drastically improve the detection of candidate genes subject to DNA methylation regulation and heterogeneously expressed among a population.…”