Continuous genetic recording with self-targeting CRISPR-Cas in human cells

Abstract

INTRODUCTION Technologies that enable the longitudinal tracking and recording of molecular events into genomic DNA would be useful for the detailed monitoring of cellular state in artificial and native contexts. Although previous systems have been used to memorize digital information such as the presence or absence of biological signals, tools for recording analog information such as the duration or magnitude of biological activity in human cells are needed. Here, we present Mammalian Synthetic Cellular Recorders Integrating Biological Events (mSCRIBE), a memory system for storing analog biological information in the form of accumulating DNA mutations in human cells. mSCRIBE leverages self-targeting guide RNAs (stgRNAs) that are engineered to direct Streptococcus pyogenes Cas9 cleavage against DNA loci that encode the stgRNAs, thus accumulating mutations at stgRNA loci as a record of stgRNA or Cas9 expression. RATIONALE The RNA-guided DNA endonuclease Cas9 introduces a double-stranded break in target DNA containing a 5′-NGG-3′ protospacer-adjacent motif (PAM) and homology to the specificity-determining sequence (SDS) of a small guide RNA (sgRNA). Once a double-strand break is introduced, the targeted DNA can be repaired via error-prone DNA repair mechanisms in human cells. We hypothesized that if a PAM sequence were introduced in the DNA locus encoding the sgRNA, the transcribed sgRNA would direct Cas9 to cleave its own encoding DNA, thus acting as a stgRNA. After error-prone repair, the mutagenized stgRNA locus should continue to be transcribed and enact additional rounds of continuous, self-targeted mutagenesis. Thus, the stgRNA locus should acquire mutations corresponding to the level of activity of the Cas9-stgRNA complex. We hypothesized that by linking the expression of stgRNA or Cas9 to biological events of interest, one could then record the duration and/or intensity of such events in the form of accumulated mutations at the stgRNA locus. The recorded information could be read by sequencing the stgRNA locus or by other related strategies. RESULTS We first built a stgRNA by engineering a sgRNA-encoding DNA locus to contain a 5′-NGG-3′ PAM immediately downstream of the SDS-encoding region. We then validated that the stgRNA could undergo multiple rounds of self-targeted mutagenesis by building a mutation-based toggling reporter system in which the progressive accumulation of mutations at the stgRNA locus is reported by individual cells toggling between green and red fluorescent protein expression. Next, we analyzed the sequence-evolution properties of stgRNAs in order to devise a sequence-based recording metric that conveys information on the duration and/or magnitude of stgRNA activity. We showed that computationally designed stgRNAs that contain longer SDSs of length 30, 40, and 70 nucleotides are able to accumulate mutations over longer durations of time. We demonstrated the analog nature of mSCRIBE by building a tumor necrosis factor–α (TNFα)–inducible Cas9 expression system and observing graded increases in the recording metric as a function of increasing TNFα concentration and/or duration of exposure in vitro . By designing doxycycline and isopropyl-β- d thiogalactoside-inducible stgRNA expression systems, we also showed inducible, multiplexed recording at two independent DNA loci. Last, we confirmed that human cells containing TNFα-responsive mSCRIBE units can record lipopolysaccharide (LPS)–induced acute inflammation events over time in mice. CONCLUSION We demonstrate that sgRNAs can be engineered to function as stgRNAs. By linking stgRNA or Cas9 expression to specific biological events of interest—such as the presence of small molecules, exposure to TNFα, or LPS-induced inflammation—we validated mSCRIBE as an analog memory device that records information about the duration and/or magnitude of biological events. Moreover, we demonstrated that multiple biological events can be simultaneously monitored by using independent stgRNA loci. We envision that this platform for genomically encoded memory in human cells should be broadly useful for studying biological systems and longitudinal and dynamic events in vitro and in situ, such as signaling pathways, gene regulatory networks, and tissue heterogeneity involved in development, healthy cell function, and disease pathogenesis. Continuously evolving stgRNAs. The Cas9-stgRNA complex cleaves the DNA locus from which the stgRNA is transcribed, leading to error-prone DNA repair. Multiple rounds of transcription and DNA cleavage can occur, resulting in progressive mutagenesis of the DNA encoding the stgRNA. The accumulation of mutations in the stgRNA locus provides a molecular record of cellular events that regulate stgRNA or Cas9 expression.