Continuous Genetic Recording with Self-Targeting CRISPR-Cas in Human Cells

Abstract

The ability to longitudinally track and record molecular events in vivo would provide a unique opportunity to monitor signaling dynamics within cellular niches and to identify critical factors in orchestrating cellular behavior. We present a self-contained analog memory device that enables the recording of molecular stimuli in the form of DNA mutations in human cells. The memory unit consists of a self-targeting guide RNA (stgRNA) cassette that repeatedly directs Streptococcus pyogenes Cas9 nuclease activity towards the DNA that encodes the stgRNA, thereby enabling localized, continuous DNA mutagenesis as a function of stgRNA expression. We analyze the temporal sequence evolution dynamics of stgRNAs containing 20, 30 and 40 nucleotide SDSes (Specificity Determining Sequences) and create a population-based recording metric that conveys information about the duration and/or intensity of stgRNA activity. By expressing stgRNAs from engineered, inducible RNA polymerase (RNAP) III promoters, we demonstrate programmable and multiplexed memory storage in human cells triggered by doxycycline and isopropyl {beta}-D-1-thiogalactopyranoside (IPTG). Finally, we show that memory units encoded in human cells implanted in mice are able to record lipopolysaccharide (LPS)-induced acute inflammation over time. This tool, which we call Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE), provides a unique strategy for investigating cell biology in vivo and in situ and may drive further applications that leverage continuous evolution of targeted DNA sequences in mammalian cells.\n\nOne Sentence SummaryBy designing self-targeting guide RNAs that repeatedly direct Cas9 nuclease activity towards their own DNA, we created multiplexed analog memory operators that can record biologically relevant information in vitro and in vivo, such as the magnitude and duration of exposure to TNA.