Research Article

Rewritable multi-event analog recording in bacterial and mammalian cells

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Science  13 Apr 2018:
Vol. 360, Issue 6385, eaap8992
DOI: 10.1126/science.aap8992

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Writing a cell's history in its DNA

Recording cellular events could advance our understanding of cellular history and responses to stimuli. The construction of intracellular memory devices, however, is challenging. Tang and Liu used Cas9 nucleases and base editors to record amplitude, duration, and order of stimuli as stable changes in both genomic and extrachromosomal DNA content (see the Perspective by Ho and Bennett). The recording of multiple stimuli—including exposure to antibiotics, nutrients, viruses, and light, as well as Wnt signaling—was achieved in living bacterial and human cells. Recorded memories could be erased and re-recorded over multiple cycles.

Science, this issue p. eaap8992; see also p. 150

Structured Abstract

INTRODUCTION

The stable recording of cellular events has the potential to advance our understanding of a cell’s history and how cells respond to stimuli. However, the construction of intracellular memory devices that record a history of cellular events has proven challenging.

RATIONALE

We developed two CRISPR-mediated analog multi-event recording apparatus (CAMERA) systems that record cellular events as durable changes in the DNA of bacteria or mammalian cells. In CAMERA 1, Cas9 nucleases are used to shift the ratio of two recording plasmids, and signals are recorded in the form of plasmid ratios. Writing in CAMERA 2 uses base editors to produce single-base modifications at designated positions of plasmid or genomic DNA. Both Cas9 nucleases and base editors can be programmed to target multiple DNA sequences with different guide RNAs, and both are known to function across many cell types. These features enable CAMERA to serve as a multiplexable, analog, rewritable intracellular recording system.

RESULTS

We demonstrate that the ratio of the recording plasmid pair in CAMERA 1 can be stably maintained in bacteria over 144 hours and a dilution ratio of 1017. By using a writing complex of the Cas9 nuclease and a guide RNA to selectively target one of the recording plasmids, we can cause this plasmid ratio to shift in a dose-dependent manner. The presence or absence of a stimulus is recorded in CAMERA 1 by linking to the expression of the writing complex. The analog format of CAMERA 1 enables recording of signal amplitude over a known time scale, or recording of the duration of a signal of known strength. Two resetting methods enable cells harboring CAMERA 1 to function over repeated cycles of recording and erasing.

CAMERA 2 uses base editors to record stimuli of interest as permanent single-base modifications in cellular DNA. Predictable and dose-dependent accumulation of base editing was observed over 68 generations in bacteria. CAMERA 2 achieved analog recording of multiple stimuli of interest, including exposure to antibiotics, nutrients, viruses, and light. When recording to a high-copy plasmid, CAMERA 2 provides reliable readout by sequencing only 10 to 100 cells and can record event order using an overlapping guide RNA design.

CAMERA 2 also functions in human cells by recording stimuli to safe-harbor genomic loci. We show that CAMERA 2 can be multiplexed, such that two responsive guide RNA expression cassettes can be used to record the presence of two exogenous small molecules in mammalian cells. Finally, we demonstrated CAMERA 2 recording of Wnt signaling, a crucial endogenous mammalian signaling pathway, as a permanent change in genomic DNA in human cells by placing the expression of the writing complex under the control of a Wnt-responsive promoter.

CONCLUSION

Base editors and CRISPR nucleases were used to create “cell data recorders” that enable durable, analog recording of stimuli and cell states. CAMERA systems are sensitive, multiplexable, resettable, and compatible with both bacteria and mammalian cells, and thus may be useful for applications such as recording the presence of extracellular and intracellular signals, mapping cell lineage, and constructing cell state maps.

Multiplexed analog cellular recording by CAMERA systems in bacteria and mammalian cells.

CAMERA 1 records stimuli as changes in the ratio of mutually exclusive DNA sequences. CAMERA 2 uses base editors to record the duration or amplitude of signals as single-nucleotide changes. Both systems can be multiplexed to independently record multiple events, including exposure to antibiotics, nutrients, viruses, and light, as well as Wnt signaling.

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.

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