Rewritable multi-event analog recording in bacterial and mammalian cells

Abstract

We present two CRISPR-mediated analog multi-event recording apparatus (CAMERA) systems that use base editors and Cas9 nucleases to record cellular events in bacteria and mammalian cells. The devices record signal amplitude or duration as changes in the ratio of mutually exclusive DNA sequences (CAMERA 1) or as single-base modifications (CAMERA 2). We achieved recording of multiple stimuli in bacteria or mammalian cells, including exposure to antibiotics, nutrients, viruses, light, and changes in Wnt signaling. When recording to multicopy plasmids, reliable readout requires as few as 10 to 100 cells. The order of stimuli can be recorded through an overlapping guide RNA design, and memories can be erased and re-recorded over multiple cycles. CAMERA systems serve as "cell data recorders" that write a history of endogenous or exogenous signaling events into permanent DNA sequence modifications in living cells.

Fig. 1. Plasmid ratios controlled by Cas9 nuclease in CAMERA 1

(A) Schematic representation of CAMERA 1 systems. Recording plasmids R1 and R2 are identical except a 3-nucleotide coding mutation in the EGFP gene. The expression of the Cas9:sgRNA complex is controlled by the signal of interest and results in R1 depletion in the bacteria that carry the recording plasmid pair. (B) Stability of the R1:R2 ratio in E. coli S1030 cells in the absence of the writing plasmid. (C) In vitro cleavage of the wild-type and mutated EGFP gene by Cas9 in the presence of sgRNA1. The designed spacer sequence targets the distinct region in EGFP so the Cas9:sgRNA complex cleaves R1 much faster than R2. (D) Recording the amplitude and duration of aTc by CAMERA 1.0. Values and error bars reflect the mean and s.d. of three replicate cultures derived from a single bacterial colony.

Fig. 2. Multi-event recording and resetting of CAMERA 1 systems

(A) Construction of a “AND” Boolean logic gate using CAMERA 1.1. Both IPTG and aTc are required for initiation of the recording process. (B) Analog recording of IPTG concentration by CAMERA 1.1 as reported by EGFP fluorescence. (C) Repeated recording and erasing of CAMERA 1.2 by application of aTc and IPTG and kanamycin. S: starting state; E: erase (5-20 generations); R: record (5-10 generations). (D) Repeated recording and erasing of CAMERA 1.3 by inducing different writing complexes. (E) Dose-dependent recording and erasing using CAMERA 1.3. Values and error bars reflect mean and s.d. of three replicates.

Fig. 3. CAMERA 2 systems use base editing to record the amplitude and duration of exogenous signals

(A) Schematic representation of CAMERA 2 systems. The writing plasmid expresses the writing complex consisting of BE2 and sgRNAs. The recording plasmid is targeted by the writing complex and generates memory in the form of C•G to T•A substitutions at guide RNA-specified loci. (B) Recording the concentration of aTc and the treatment duration in analog mode using CAMERA 2.0. (C) Recording the concentration of IPTG in the presence or absence of aTc and the treatment duration in analog mode using CAMERA 2.1. (D) The rate of base editing at position 166 of the EGFP gene in CAMERA 2.0 reflects the schedule of exposure to the inducer. (E) CAMERA 2.1 records the total time of exposure to IPTG, regardless of treatment pattern. (F) Recording four exogenous stimuli using CAMERA 2.4. The presence of each signal, individually or in combinations, was recorded by base editing at each of several specified positions in the EGFP gene. We constructed two mathematical models to simulate the behavior of CAMERA 2.4. A model that accounts for promoter leakage and competition between multiple guide RNAs for BE2 results in a more “digital” CAMERA 2.4 in which the absolute editing level at each position more readily reveals the presence or absence of the corresponding stimulus (fig. S7). Values and error bars reflect the mean and s.d. of three replicates.

Fig. 4. CAMERA 2 systems can record the order of stimuli and a wide range of environmental signals

(A) Schematic representation of CAMERA 2.5 that records stimuli in an order-dependent manner. (B) CAMERA 2.6 records the presence of arabinose at position 205-207 in format of C•G to T•A mutations. (C) The ratio of base editing at position 216:position 129 in CAMERA 2.5 indicates the order of exposure to two stimuli. A position 216:129 base editing ratio above 0.1 was only observed when the bacteria were treated first with arabinose and then with rhamnose, but not if arabinose exposure follows rhamnose exposure. (D) Phage infection recording by CAMERA 2.6. (E) Light exposure recording with CAMERA 2.7 in bulk culture and in small numbers of cells. Light exposure duration can be recorded faithfully in bulk culture as well as in samples of only 100 or 10 cells. Values and error bars in bar graphs reflect the mean and s.d. of three replicates. Dots and error bars in dot plots in (E) represent the mean and s.d. of 15 replicates of randomly sorted sets of many, 100, or 10 cells.

Fig. 5. CAMERA 2m recordings in mammalian cells

(A) Schematic representation of CAMERA 2m in mammalian cells. (B) CAMERA 2m.0 functions in a multiplexed manner in mammalian cells by targeting the human safe harbor gene CCR5. (C) CAMERA 2m.1 records the presence of doxycycline in HEK293T cells through a doxycycline-controlled transcriptional activator. (D) CAMERA 2m.2 records the presence of doxycycline and IPTG in a multiplexed manner. Expression of sgRNA A and sgRNA B is repressed by LacI and TetR in the absence of stimuli and can be turned on at the addition of IPTG and doxycycline, respectively. (E) CAMERA 2m.3 responds to Wnt signaling and records the presence of a Wnt signaling stimulus at a target genomic safe harbor locus. Values and error bars reflect mean editing and the s.d. of three replicates.