Ateam of Swiss scientists has engineered bacteria that are capable of making indelible DNA accounts of the most abundant RNAs the cells produce, according to a report in Nature earlier this month (October 3). The developers used the organisms in proof-of-principle experiments to record the cells’ transcriptional responses to both stress and a common herbicide.
“They’ve co-opted the natural way that the CRISPR system works—by storing information from nucleic acids that are invading hosts—[and used it] to capture events that are actually going on inside the cells,” says genomics researcher John Stamatoyannopoulos of the University of Washington in Seattle who was not involved in the research. “It’s a clever application.”
The natural CRISPR-Cas system is a bacterial immune mechanism that inserts into the host genome short pieces of DNA (called spacers) cut from invading viruses. These DNA spacers—like dossiers of criminals on a most-wanted list—can later be used for recognition and destruction of the viruses should any of them return.
Because of the predictable, nonrandom nature of spacer acquisition and integration, scientists have been able to repurpose the mechanism to their own ends, using it to keep tabs on the presence of other forms of extrinsic DNA. Biological engineer Randall Platt and colleagues of the Swiss Federal Institute of Technology in Zurich have now gone one step further, using the CRISPR system to collect and record pieces of cellular RNA.
RNA-seq is like taking pictures, and [Record-seq] is like taking movies.
For this, the team took advantage of a naturally occurring version of the Cas1 protein—the enzyme that snips spacers from invading viral genomes—that has reverse transcriptase (RT) activity, the ability to turn RNA into DNA. This RT-Cas1 enzyme can thus both excise pieces of cellular RNAs and convert them into DNA. More than 100 such RT-Cas1 proteins exist in nature, though their role in bacterial defense, if any, is not entirely clear. Platt and colleagues tested all RT-Cas1 proteins they could identify before finding just one (from Fusicatenibacter saccharivorans) that was capable of converting RNA snippets into DNA spacers when expressed in E coli.
“RNA generally is transient and it degrades very quickly, but they were able to essentially utilize the RT-Cas system to permanently record that information in the genome,” says systems biologist Harris Wang of Columbia University Medical Center in New York who was not part of the research team. “That allows you to record [transcriptional] information that is happening in real time.”
By transfecting E. coli with a plasmid containing the F. saccharivorans RT-Cas1 gene and a DNA motif (a repeat sequence) used by the CRISPR-Cas machinery for spacer insertion, the team could in principle press record (induce RT-Cas1 expression), expose the cells to assorted environments, and then subsequently sequence the spacer-containing CRISPR locus to read the record of any RNAs expressed by the cell. Because spacer elements are linearly ordered within the DNA with respect to the temporal order in which they were acquired, sequencing the CRISPR loci would, in theory, reveal not only the RNAs expressed, but their order of expression.
As yet, however, the approach—which the researchers call Record-seq—is quite inefficient, says Platt, with only roughly one spacer acquisition event per 20,000 E. coli cells. The spacers acquired therefore represent only the most abundantly expressed RNAs and provide little if any temporal information.
Nevertheless, the approach was sufficiently robust that the team was able to detect RNAs upregulated in a bacterial population as a result of stress—caused by treatment with hydrogen peroxide or acid—and transient exposure to the common herbicide paraquat.
The team also performed whole transcriptome sequencing—a technique called RNA-seq—of the bacteria. In initial experiments, they used this to validate the findings of Record-seq, but they also showed that, after transient paraquat exposure, when culture conditions had returned to normal, Record-seq was able to retrieve the bacterial record of genes transiently upregulated by the herbicide, while RNA-seq could not.
That’s because “RNA-seq is like taking pictures, and [Record-seq] is like taking movies,” says MIT’s Fahim Farzadfard, a postdoc in the lab of Ed Boyden who did not participate in the research. While the Record-seq approach will need further improvement, he continues, “it’s a very exciting development in molecular recording technology . . . [and] a new way of thinking about how we can study transcription.”
F. Schmidt et al., “Transcriptional recording by CRISPR spacer acquisition from RNA,” Nature, 10.1038/s41586-018-0569-1, 2018.