The CRISPR-Cas system, while originally hailing from the Domain of Bacteria, has made the biggest splashes by traveling abroad into the Kingdoms of Plantae and Animalia. There it has found great fame and fortune mostly as a tool for genome editing. Scientists back in the Bacterial Domain didn’t need CRISPR as much for gene editing, because they already had plenty of tools for that. But as CRISPR’s fame has grown, synthetic biologists have realized they could also harness its sequence-specificity to make dynamic genetic circuits.
One key challenge in synthetic biology has been the limited number of “parts” – genetic switches that can be turned on or off by other genes with high specificity. Several labs have shown how to use a non-cutting Cas9 as a highly targeted transcriptional switch. Extending this to translational control, a new paper from Chunbo Lou’s lab shows how CRISPR can be used to activate genes post-transcriptionally in E. coli.
Post-Transcriptional Activation with Cas6
In this approach, first author Pei Du fused a cis-repressing RNA (crRNA, not to be confused with the equally-acronymed CRISPR targeting RNA) to the 5’ UTR of GFP. The crRNA pairs with the ribosome-binding site (RBS), thus blocking translation. Next, the team stuck a palindromic repeat (putting the PR into CRISPR) between the crRNA and the RBS. In a normal CRISPR system, RNA with these repeats is recognized and cut by Cas6 homologs to separate and release the individual DNA-targeting spacer regions. In this case, cutting the spacer un-tethers the crRNA from the RBS, thus relieving translation repression.
In their first iteration, the team got 39-fold activation of GFP when they induced the Cas6 homolog Csy4. Even after activation, though, the expression still wasn’t as high as in a control without the crRNA. Adding some mismatches within the crRNA increased induction to 63-fold, but at the cost of increased background. The team also tried varying the length of the crRNA, but didn’t find a clear pattern for inducibility.
Multiplexing CRISPR Translational Activation
While this system does use CRISPR, it doesn’t really take full advantage of CRISPR’s sequence-specificity, since Csy4 will cleave only one specific repeat sequence and can’t be retargeted. However, the team tested several Cas6 homologs and found 3 that work in their system and are relatively orthogonal. Using this system, then, gene circuit designers now have 3 new translationally activating switches to play with.
For more, check out the paper at ACS Syn Bio, 2015.