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. Here, we use CRISPR spacer acquisition to capture and convert intracellular RNAs into DNA, enabling DNA-based storage of transcriptional information. In Escherichia coli, we show that defined stimuli, such as an RNA virus or arbitrary sequences, as well as complex stimuli, such as oxidative stress, result in quantifiable transcriptional records that are stored within a population of cells. We demonstrate that the transcriptional records enable us to classify and describe complex cellular behaviours and to identify the precise genes that orchestrate differential cellular responses. In the future, CRISPR spacer acquisition-mediated recording of RNA followed by deep sequencing (Record-seq) could be used to reconstruct transcriptional histories and thereby that describe complex cell behaviours or pathological states.A central challenge in biology is to understand how the molecular components of a cell function and integrate to enable complex cell behaviours. This challenge has fuelled the creation of increasingly sophisticated technologies that facilitate detailed intracellular observations at the levels of DNA, RNA, protein, and metabolites 1 . In particular, RNA sequencing technologies enable researchers to quantify transcriptomes within multiple or single cells, revealing the molecular signatures of cell behaviours, states, and types with unprecedented detail 2,3 . Despite the power of these technologies, they require destructive methods and therefore observations are limited to a few snapshots in time or to select asynchronous cellular processes. One provocative solution to this is to introduce synthetic memory devices 4 within cells that enable the encoding, storage, and retrieval of transcriptional information.Supplementary Table 2). Using a previously established spacer acquisition assay 27 , we found that only one of the orthologues tested (F. saccharivorans) actively acquired new spacers (Extended Data Fig. 1c). The endogenous F. saccharivorans locus contains two CRISPR arrays and we observed that novel spacers derived from the overexpression plasmid as well as the E. coli genome were acquired into either array (Extended Data Fig. 1c-e). Selective amplification of expanded CRISPR arraysUsing the previously established spacer acquisition assay 27 , we obtained approximately 1,300 newly acquired spacers per 1 million deep sequencing reads for FsRT-Cas1-Cas2 (Extended Data Fig. 1c). To improve detection of novel spacers, we developed 'selective amplification of expanded CRISPR arrays' (SENECA), a method for selective amplification of CRISPR arrays that acquired new spacers (Fig. 2a, Extended Data Fig. 2a). A typical SENECA-assisted Recordseq experiment uses an input of about 180 ng plasmid DNA extracted from an overnight culture of E. coli overexpressing...

Leukemia inhibitory factor (LIF) is indispensable to maintain the pluripotent state of mouse embryonic stem cells (ESCs), but the mechanisms underlying the role of LIF/STAT3 pathway are yet poorly understood. Here we first showed that the LIF/STAT3-regulated signaling pathway contributes to the maintenance of self-renewal and pluripotency of mouse ESCs by suppressing mTOR (mammalian target of rapamycin), which is necessary for early differentiation. When LIF is withdrawn from culture medium, the mTOR activity rapidly increases as detected by phosphorylation of its targets – ribosomal protein S6 and translation factor 4EBP1. In turn, suppression of STAT3 phosphorylation on Tyr-705 by a specific small molecule WP1066 also activates phosphorylation of the mTOR target S6 ribosomal protein. LIF removal strongly activates ERK activity indicating that ERK can be involved in either direct phosphorylation of mTOR or phosphorylation of an upstream negative regulator of mTOR – TSC1/TSC2 proteins. According to western blotting data, LIF withdrawal leads to phosphorylation of TSC2 protein thereby relieving its negative effect on mTOR activity. mTOR activation is accompanied by a decrease of pluripotent gene expression Oct-4, Nanog, Sox2 and by an augmentation of fgf5 gene expression – a marker of post-implantation epiblast. Together, these data indicate that LIF-depleted mouse ESCs undergo a transition from the LIF/STAT3-supported pluripotent state to the FGFR/ERK-committed primed-like state with expression of early differentiation markers mediated through activation of mTOR signaling.

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...

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