Scientists have harnessed the bacterial CRISPR/Cas immune system to generate miniature living tape recorders that could feasibly be used to diagnose disease, carry out environmental or microbiological sensing, and record changes in body organs or systems such as the gut.
“Such bacteria, swallowed by a patient, might be able to record the changes they experience through the whole digestive tract, yielding an unprecedented view of previously inaccessible phenomena,” comments Harris Wang, Ph.D., assistant professor in the department of pathology and cell biology and systems biology at Columbia University Medical Center (CUMC), who led the research. Dr. Wang is also senior author of the group’s published paper in Science, which is entitled “Multiplex Recording of Cellular Events Over Time on CRISPR Biological Tape.”
The bacterial CRISPR/Cas system exemplifies a naturally occurring biological memory system, the authors explain. When a bacterium is infected by a genetic element, such as a phage or a plasmid, the cell’s CRISPR/Cas system cuts out short fragments of the foreign nucleic acid and integrates the sequence into genomic CRISPR arrays as spacers. The CRISPR/Cas immunity proteins can then recognize and eliminate the same invader if it tries to infect the bacterium a second time, at any point in the future. Importantly, the integration of foreign DNA occurs unidirectionally, which means that the CRISPR locus represents a chronological record of invading viruses that is passed down through bacterial generations.
“The CRISPR/Cas system is a natural biological memory device,” says Wang. “From an engineering perspective, that's actually quite nice, because it's already a system that has been honed through evolution to be really great at storing information.”
Tape recorders convert temporal signals, such as analog audio, into recordable data written on the tape as it passes through a recorder at a set speed. The researchers aimed at developing a similar biological system, which they call temporal recording in arrays by CRISPR expansion (TRACE). “When you think about recording temporally changing signals with electronics, or an audio recording…that's a very powerful technology, but we were thinking how can you scale this to living cells themselves?” notes Ravi Sheth, a graduate student in Wang’s laboratory.
CRISPR has previously been used to record and store poems, books, and even an experimental movie in DNA, but this is the first time CRISPR has been used to record cellular activity and the timing of those events. The authors report “…engineering the CRISPR-Cas adaptation system to directly record biological signals and their temporal context, and not simply sequence information of exogenous DNA, has not been achieved to-date.… In this framework, a biological input signal is first transformed into a change in the abundance of a trigger DNA pool within living cells. The CRISPR-Cas spacer acquisition machinery is then employed to record the amount of trigger DNA into CRISPR arrays in a unidirectional manner.”
To generate their bacterial recording system the researchers engineered two different plasmids in a laboratory strain of Escherichia coli. The first plasmid created more copies of itself in response to an external signal. A second recording plasmid effectively marked time and expressed the required CRISPR/Cas system components. When there was no external signal, the recording plasmid continued to insert copies of a spacer sequence into the CRISPR locus. When an external signal was detected, however, the self-replicating plasmid was activated, leading to insertion of signal sequences. What you get is a background of spacer sequences that follow time, interspersed with signal sequences that were inserted as a result of changes in the cell’s environment. The CRISPR locus can then be read using computational tools. Initial tests with the TRACE system showed that recorded information was stable within cell populations over eight days.
Having generated their prototype system, the team then demonstrated how it could be multiplexed, enabling the simultaneous recording of three different signals—in this case availability of the metabolites copper, trehalose, and fucose—in the cell population environment over three days. “This work enables the temporal measurement of dynamic cellular states and environmental changes and suggests new applications for chronicling biological events on a large scale,” the authors conclude. “Our work enables new applications in biological recording. TRACE could be utilized to record metabolite fluctuations, gene expression changes, and lineage-associated information across cell populations in difficult-to-study habitats such as the mammalian gut or in open settings such as soil or marine environments.”
Dr. Wang says the team next plans to use TRACE platform to investigate markers that indicate changes in normal or diseased states in the gastrointestinal tract and other body systems.