The CRISPR systems inside bacteria serve as adaptive immune systems, but they also threaten to unleash autoimmune reactions. Fortunately, for bacteria such as Streptococcus pyogenes, these systems have a built-in safety feature: a long-form transactivating CRISPR RNA (tracrRNA). Unlike the short-form tracrRNA, which together with CRISPR RNA (crRNA) complexes with the CRISPR-Cas9 enzyme and guides it to DNA sites where it executes cuts, the long-form tracrRNA guides the enzyme to the enzyme’s own genetic promoter.

The short-form tracrRNA that complexes CRISPR-Cas9 enzyme doesn’t need to bind to crRNA, and it doesn’t cut. Instead, it merely lingers in place, preventing gene expression.

Essentially, long-form tracrRNA acts as a safety feature, dialing down a bacterium’s immune system to prevent it from attacking the bacterium itself rather than foreign DNA. This self-protection function for long-form tracrRNA was uncovered by researchers at Johns Hopkins University. The researchers, led by Joshua W. Modell, PhD, also explored whether long-form tracrRNA could be reprogrammed to guide CRISPR-Cas9 to DNA sites other than the CRISPR-Cas9 promoter.

The researchers’ findings appeared January 8 in the journal Cell, in an article titled, “A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression.” According to the researchers, long-form tracrRNA could serve as a programmable genetic dimmer switch, one that could be used to inhibit the expression of designated genes in research applications.

“We show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas),” the article’s authors wrote. “Further, we demonstrate that Pcas serves as a critical regulatory node.”

Scientists have long worked to unravel the precise steps of CRISPR-Cas9’s mechanism and how its activity in bacteria is dialed up or down. Looking for genes that ignite or inhibit the CRISPR-Cas9 gene-cutting system for the common, strep-throat causing bacterium S. pyogenes, the Johns Hopkins scientists found a clue regarding how that aspect of the system works.

Specifically, the scientists found a gene in the CRISPR-Cas9 system that, when deactivated, led to a dramatic increase in the activity of the system in bacteria. The product of this gene appeared to re-program Cas9 to act as a brake, rather than as a “scissor,” to dial down the CRISPR system.

“From an immunity perspective, bacteria need to ramp up CRISPR-Cas9 activity to identify and rid the cell of threats, but they also need to dial it down to avoid autoimmunity—when the immune system mistakenly attacks components of the bacteria themselves,” said graduate student Rachael Workman, a bacteriologist working in Modell’s laboratory.

To further nail down the particulars of the “brake,” the team’s next step was to better understand the product of the deactivated gene, a tracrRNA. tracrRNAs belong to a unique family of RNAs that do not make proteins. Instead, they act as a kind of scaffold that allows the Cas9 enzyme to carry the guide RNA that contains a “mug shot” of previously encountered phage DNA. The mug shot allows Cas9 to cut matching DNA sequences in newly invading viruses.

tracrRNA comes in two sizes: long and short. Most of the modern gene-cutting CRISPR-Cas9 tools use the short form. However, the research team found that the deactivated gene product was the long form of tracrRNA, the function of which has been entirely unknown.

 

In bacteria, DNA-cutting CRISPR-Cas9 complexes typically consist of a Cas9 enzyme and a guide RNA. The guide RNA consists of a short-form transactivating CRISPR RNA (tracrRNA) scaffold and a DNA-sequence-specific CRISPR (crRNA). Long-form tracrRNA, however, can complex with and guide Cas9 without crRNA. When long-form tracrRNA does so, it guides the Cas9 enzyme to a Cas9 promoter. The promoter is not cut, but expression is repressed. Left: A schematic of the long-form of the tracrRNA used by the CRISPR-Cas9 system in bacteria. Right: the standard guide RNA used by many scientists as part of the gene-cutting CRISPR-Cas9 system. (Often, the guide RNA is a single synthetic molecule, rather than a combination of tracrRNA and crRNA.) [Joshua Modell and Rachael Workman, Johns Hopkins Medicine]

The long and short forms of tracrRNA are similar in structure and have in common the ability to bind to Cas9. The short-form tracrRNA also binds to the guide RNA. However, the long-form tracrRNA doesn’t need to bind to the crRNA, because it contains a segment that mimics the crRNA. Essentially, long-form tracrRNAs have combined the function of the short-form tracrRNA and crRNA, explained Modell, assistant professor of molecular biology and genetics at the Johns Hopkins University School of Medicine.

The researchers used genetic engineering to alter the length of a certain region in long-form tracrRNA to make the tracrRNA appear more like a guide RNA. They found that with the altered long form tracrRNA, Cas9 once again behaved more like a scissor.

Other experiments showed that in lab-grown bacteria with a plentiful amount of long-form tracrRNA, levels of all CRISPR-related genes were very low. When the long-form tracrRNA was removed from bacteria, however, expression of CRISPR-Cas9 genes increased a hundredfold.

Bacterial cells lacking the long-form tracrRNA were cultured in the laboratory for three days and compared with similarly cultured cells containing the long-form tracrRNA. By the end of the experiment, bacteria without the long-form tracrRNA had completely died off. “De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses,” the article’s authors noted. “However, heightened immunity comes at the cost of increased autoimmune toxicity.”

These findings suggest that long-form tracrRNA normally protects cells from the sickness and death that happen when CRISPR-Cas9 activity is very high. “We started to get the idea that the long form was repressing but not eliminating its own CRISPR-related activity,” recalled Workman.

To see if the long form tracrRNA could be re-programmed to repress other bacterial genes, the research team altered the long form tracrRNA’s spacer region to let it sit on a gene that produces green fluorescence. Bacteria with this mutated version of long-form tracrRNA glowed less green than bacteria containing the normal long form tracrRNA, suggesting that the long-form tracrRNA can be genetically engineered to dial down other bacterial genes.

Another research team, from Emory University, found that in the parasitic bacteria Francisella novicida, Cas9 behaves as a dimmer switch for a gene outside the CRISPR-Cas9 region. The CRISPR-Cas9 system in the Johns Hopkins study is more widely used by scientists as a gene-cutting tool, and the Johns Hopkins team’s findings provide evidence that the dimmer action controls the CRISPR-Cas9 system in addition to other genes.

“Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A CRISPR-Cas systems,” the Johns Hopkins scientists added. “Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.”

The researchers also found the genetic components of long-form tracrRNA in about 40% of the Streptococcus group of bacteria. Further study of bacterial strains that don’t have the long-form tracrRNA, said Workman, will potentially reveal whether their CRISPR-Cas9 systems are intact, and other ways that bacteria may dial back the CRISPR-Cas9 system.

The dimmer capability that the experiments uncovered offers opportunities to design new or better CRISPR-Cas9 tools aimed at regulating gene activity for research purposes. “In a gene editing scenario,” Modell suggested, “a researcher may want to cut a specific gene, in addition to using the long-form tracrRNA to inhibit gene activity.”

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