Biological engineers at MIT have developed a new way to efficiently edit bacterial genomes and program memories into bacterial cells by rewriting their DNA. Using this approach, various forms of spatial and temporal information can be permanently stored for generations and retrieved by sequencing the DNA of cells.
The new DNA-writing technique, which the researchers call HiSCRIBE, is much more efficient than previously developed systems for DNA editing in bacteria, which had a success rate of about 1 cell in 10,000 per generation only. In a new study, the researchers demonstrated that this approach could be used to store memory of cellular interactions or spatial location.
This technique could also allow genes to be selectively edited, activated or silenced in certain species of bacteria living in a natural community such as the human microbiome, according to the researchers.
“With this new DNA writing system, we can accurately and efficiently edit bacterial genomes without the need for any form of selection, within complex bacterial ecosystems,” says Fahim Farzadfard, a former postdoctoral fellow at MIT and main author of the article. “This allows us to perform genome editing and DNA writing outside of laboratory settings, whether to engineer bacteria, optimize traits of interest in situ, or study evolutionary dynamics and interactions in bacterial populations.”
Timothy Lu, an associate professor of electrical engineering and computer science and biological engineering at MIT, is the lead author of the study, which appears today in Cell systems. Nava Gharaei, a former Harvard University graduate student, and Robert Citorik, a former MIT graduate student, are also the authors of the study.
Writing the genome and recording memories
For several years, Lu’s lab has been working on ways to use DNA to store information such as the memory of cellular events. In 2014, he and Farzadfard developed a way to employ bacteria as a “genomic tape recorder,” engineering E.coli to store long-term memories of events such as chemical exposure.
To achieve this, the researchers engineered the cells to produce a reverse transcriptase enzyme called retron, which produces single-stranded DNA (ssDNA) when expressed in cells, and a recombinase enzyme, which can insert (“write”) a specific single-stranded DNA sequence at a targeted site in the genome. This DNA is only produced when it is activated by the presence of a predetermined molecule or another type of input, such as light. Once the DNA is produced, the recombinase inserts the DNA into a pre-programmed site, which can be anywhere in the genome.
This technique, which the researchers called SCRIBE, had relatively low writing efficiency. At each generation, out of 10,000 E.coli cells, only one acquired the new DNA that the researchers tried to incorporate into the cells. This is partly because the E.coli have cellular mechanisms that prevent single-stranded DNA from accumulating and integrating into their genomes.
In the new study, the researchers attempted to increase the efficiency of the process by eliminating some of the E.coli’s defense mechanisms against single-stranded DNA. First, they disabled enzymes called exonucleases, which break down single-stranded DNA. They also knocked out genes involved in a system called mismatch repair, which normally prevents single-stranded DNA from integrating into the genome.
With these modifications, the researchers were able to achieve near-universal incorporation of the genetic modifications they attempted to introduce, creating an unprecedented and efficient way to edit bacterial genomes without the need for selection.
“Thanks to this improvement, we were able to do some applications that we weren’t able to do with the previous generation of SCRIBE or with other DNA writing technologies,” says Farzadfard.
In their 2014 study, the researchers showed that they could use SCRIBE to record the duration and intensity of exposure to a specific molecule. With their new HiSCRIBE system, they can trace these types of exposures as well as other types of events, such as interactions between cells.
As an example, the researchers showed they could track a process called bacterial conjugation, in which bacteria swap pieces of DNA. By embedding a DNA “barcode” into each cell’s genome, which can then be swapped with other cells, researchers can determine which cells have interacted with each other by sequencing their DNA to see which barcodes they carry.
This type of mapping could help researchers study how bacteria communicate with each other within aggregates such as biofilms. If a similar approach could be deployed in mammalian cells, it could one day be used to map interactions between other cell types such as neurons, Farzadfard says. Viruses that can cross neural synapses could be programmed to carry DNA barcodes that researchers could use to trace connections between neurons, offering a new way to help map the brain’s connectome.
“We use DNA as a mechanism to record spatial information about the interaction of bacterial cells, and perhaps in the future, neurons that have been labeled,” says Farzadfard.
The researchers also showed that they could use this technique to specifically edit the genome of a species of bacteria within a multi-species community. In this case, they introduced the gene for an enzyme that breaks down galactose into E.coli cells growing in culture with several other species of bacteria.
This kind of species-selective editing could offer a new way to make antibiotic-resistant bacteria more susceptible to existing drugs by silencing their resistance genes, the researchers say. However, such treatments would likely require several more years of research to develop, they say.
The researchers also showed that they could use this technique to design a synthetic ecosystem composed of bacteria and bacteriophages capable of continuously rewriting certain segments of their genome and evolving autonomously at a rate greater than that which would be possible by natural evolution. In this case, they were able to optimize the cells’ ability to consume lactose consumption.
“This approach could be used for evolutionary engineering of cellular traits or in experimental evolutionary studies by allowing you to replay the evolutionary tape over and over again,” says Farzadfard.
The research was funded by the National Institutes of Health, Office of Naval Research, National Science Foundation, Defense Advanced Research Projects Agency, MIT Center for Microbiome Informatics and Therapeutics, NSF Expeditions in Computing Program Award, and Schmidt Science Scholars Program.