Earlier this year, Chris Voigt’s lab wrote an automated composer for synthetic gene circuits, which can design circuits to do what you want, all the way down to the DNA sequence. As good musicians know, however, the work doesn’t stop once the composer’s ink dries on the page – the next step is to find a good conductor. To address that, scientists headed by Weiren Huang and Zhiming Cai at Shenzhen University, China have been hard at work developing a CRISPR-based “signal conductor” for gene circuits.
CRISPR/Cas9 has shown impressive chops as a regulator of gene expression, but linking regulation to the presence of specific signals is a tougher problem. The new design makes three significant contributions for conducting information flow from signals to gene expression:
- Controlling gene expression with a small molecule-sensing RNA aptamer
- Using different Cas9 variants in the same cell, and
- Creating all basic Boolean logic gates.
RNA Aptamer / CRISPR sgRNA Hybrids
To enable Cas9 to react to small molecule signals, the team developed a new “signal conductor” RNA molecule, containing a signal-binding RNA aptamer in addition to the usual Cas9-binding sgRNA scaffold and target-binding guide RNA sequence. In this expanded sgRNA, a complementary sequence between the scaffold and aptamer domains hides the guide sequence by base-pairing with it. When the aptamer domain binds its ligand, however, the whole RNA molecule changes conformation, releasing the guide sequence to go find its DNA target. If the cell is expressing nuclease dead dCas9, this will repress the target gene by steric hindrance, but if the cell is expressing dCas9-VP64, the conductor will activate transcription.
In principle, fusing any RNA aptamer to the sgRNA should work, and the authors have already tested aptamer domains that bind tetracycline, theophylline, the bacteriophage coat protein MS2, β-catenin, NF-κB, REV, and the cancer-related proteins p53, HSF-1, AFP, and NPM. In various different designs, the activation or repression efficiency was around 10-20x.
Combining dCas9 Repression And dCas9-VP64 Activation In The Same Cell
Cas9 can be made into either a gene repressor (dCas9) or an activator (dCas9-VP64). The two versions are tough to combine in the same cell though, because they recognize the same sgRNA scaffold. Overcoming this dissonance, the team used their aptamer/sgRNA conductors to make the pair play in harmony, by fusing one modified Cas9 to MS2 and the other to Rev. Then they put RNA aptamers binding either MS2 or Rev in place of key stem-loops in sgRNA scaffolds. This means instead of binding the now-missing sgRNA scaffold, each modified Cas9 will only bind “signal conductor” sgRNAs containing its complementary aptamer – either MS2 or Rev.
CRISPR-Based Boolean Logic
In a final clever little Rondo, the team showed how sgRNAs with different activation/repression strengths can be combined to create all two-input Boolean logic gates:
- AND: two weakly activating sgRNAs that synergistically become much stronger when combined
- NAND: same as AND, but with a repressing dCas9
- OR: two strongly activating sgRNAs
- NOR: two strongly repressing sgRNAs
- XOR: two strongly activating sgRNAs that target the same site on opposite DNA strands, so they base-pair with and neutralize each other if expressed simultaneously
- XNOR: same as XOR, but with a repressing dCas9
If your gene circuits could stand to be a bit more melodious, definitely check out these new CRISPR signal conductors in Nature Methods, 2016.