Turning gene expression up and down is a powerful use of CRISPR/Cas9. Labs around the world have been hard at work figuring out how to use CRISPR to control gene expression – including both transcriptional activation and repression, and then extending it to control translation and the epigenome. Cas9 can be light-activated, and with some clever use of small molecule-binding RNA aptamers, activating and repressing Cas9 variants can be used in the same cell at the same time, paving the way for CRISPR logic gates.
Now, a new paper shows an alternative approach for combining inducible activation and repression in the same cell. This paper, from Yuchen Gao and Xin Xiong, in Wendell Lim and Lei Qi’s labs at UCSF and Stanford, uses inducible dimerization. Instead of splitting deactivated Cas9 (dCas9), they put complementary dimerizing partners on dCas9 and its effector: either VPR, for activation, or KRAB, for repression.
Chemically Inducible CRISPR Gene Regulation
The dimerized labs first screened six chemical- and light-inducible dimerization systems in HEK293T cells, identifying the best as ABI-PYL1, induced by abscisic acid (ABA) and GID1-GAI, induced by gibberellin (GA). Of note for those trying to reproduce this, it was apparently important which dimerizing domain was attached to Sp dCas9 and which was on the activation/repression domain.
Inducible systems are cool not only because you can turn them on at will, but you can also dial them up gradually, and you can reverse them by removing the inducer. Cas9 activation induced by either ABA or GA was reversible on a ~1 day time scale in HEK293T cells, and both inducers had a (population-level) linear response covering at least a 10-fold range of inducer concentration.
Two Orthogonal, Inducible Cas9s For Combinatorial Regulation
With 2 chemical induction systems, the team needed a second, orthogonal dCas9 to allow each inducer to control a different set of genes. For that, they turned to Staph. aureus (Sa) dCas9. While Sa dCas9 only turned up GFP expression about 25% as much as Sp dCas9, it still achieved a 20-40x GFP increase.
With two separately inducible dCas9’s, the team could then test their key question: are the systems truly orthogonal, or do they have crosstalk? Fortunately, both the chemical inducers and the sgRNAs were inducible, meaning this system can controllably control expression of two sets of target genes.
Finally, they sorted out some combinatorial logic gates. For an OR gate, they fused the two dimerization domains onto either end of the same spCas9, linking both complementary domains to gene-activating VPR. For an AND gate, they placed the two domains in tandem. By replacing VPR with the gene-repressing KRAB, they could turn these inducible Cas9s into NOR and NAND gates. They did try placing inducible activation and repression on the same Cas9, but this regulator had a very small dynamic range.
If we’ve induced you to try and dimerize this technique and your own experiment, you can find more at Nature Methods, 2016.