We specialize in developing technologies in the space of genome editing and bifunctional molecules, and applying those technologies to beta-cell biology.
CRISPR-associated nucleases (e.g., Cas9) are programmable RNA-guided nuclease that continue to furnish disruptive technologies, including those for genome-, epigenome- and transcriptome-editing, and transcription activation/repression. Precision control lies at the heart of powerful technologies and CRISPR-based technologies are no exceptions—they require multidimensional control over half-life, dosage, and spatiotemporal (See review and TEDMED talk here). We develop CRISPR controllers (activators, inhibitors, and degraders) to elevate these technologies to their fullest potential, pivoting on specific applications in beta-cell engineering (see below).
With the innumerable negative consequences of elevated and prolonged activity of Cas9 in a cell (e.g., off-target editing, genotoxicity), many applications would benefit from a default switched-off state for Cas9-based technologies can be activated at precise times and with dose control. Our lab has developed chemical and photo-chemical activators to control Cas9-based technologies in cells and organisms (e.g., gene drives).
(Nat Chem Biol 2017). We fused Cas9 to destabilized domains that are largely unfolded and targeted, along with Cas9, for rapid proteasomal degradation. Addition of small molecule stabilizes these domains and rescues the fusion protein from degradation. By using this engineered Cas9 system, we were able to rapidly, reversibly, and dose-dependently control both genome editing and transcription technologies of Cas9.
(Angew Chem Int Ed 2019). High-resolution, non-invasive, spatiotemporal control of Cas9 activity can be achieved using light. We photocaged our small-molecule activator to afford a singular system with precise dose and spatiotemporal control of Cas9 activity. This system is rapidly responsive, requires low light intensity and exposure times, and provides reversible and multi-wavelength control of Cas9.
(Cell Reports 2020). CRISPR-based gene drives have the potential to fight vector-borne diseases or suppress crop pests because they can rapidly transmit important genes through a population. They do this by surpassing the 50% inheritance limit of Mendel’s law of independent assortment through their gene-editing technology that converts a wildtype allele to a gene-drive one. However, contemporary gene drives could spread through populations to permanently modify the genome of organisms, posing safety concerns that limit their use in both laboratory and field settings if left unchecked. We describe in Drosophila the first gene-drive system controlled by an engineered Cas9 and a synthetic, orally-available small molecule, which can be applied to field applications.
The identification of selective inhibitors of the different Cas nucleases could help improve the efficacy of gene therapies and reduce the risks associated with the off-target interactions. The identification of small-molecule inhibitors of Cas nucleases is challenging for multiple reasons. First, inhibitor identification requires robust, orthogonal, sensitive, high-throughput, miniature, and inexpensive Cas9 assays, which were unavailable. Second, Cas9 is a single turnover enzyme that holds on to its substrate with picomolar affinity throughout the biochemical reaction, adding to the challenge of developing such high-throughput assays. Third, the inhibition of Cas9 activity requires the inactivation of two nuclease domains (HNH and RuvC). Fourth, Cas9 possesses novel protein folds, limiting the ability to leverage existing rational design approaches. Fifth, Cas9 is a DNA-binding protein, a class of targets that are often deemed chemically intractable. Finally, different classes of Cas nucleases also exhibit wildly different operating mechanisms, which could make it challenging to translate assays between systems. We have resolved these issues to report first examples of cell-permeable, synthetic anti-CRISPRs.
(Chemical Sciences 2019) We have developed a sensitive, high-throughput, cell-free method wherein Cas nuclease activity is amplified via an in vitro transcription reaction that produces a fluorescent RNA:small-molecule adduct.
We created a generalizable platform that provided the first synthetic inhibitors of Cas9 that weigh <500 Da and are cell-permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for Cas9 functions, including a primary screening assay for Cas9: DNA interaction, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify inhibitors. Using these synthetic anti-CRISPRs small molecules, we demonstrated dose and temporal control of Cas9 and catalytically impaired Cas9-based technologies, including transcription activation.
Several next-generation CRISPR-associated nucleases have emerged (e.g., SaCas9, Cas12a) with superior attributes. For example, SaCas9 is much smaller than SpCas9.19 While SpCas9 catalyzes blunt cuts, Cas12a leaves sticky ends. Cas12a employs smaller guide RNA and also can self-process an RNA sequence containing multiple gRNA target sequences into multiple gRNAs, thereby allowing easy multiplex genome editing. We have developed a suite of high-throughput assays for SpCas9, SaCas9, and Cas12a and performed small-molecule screening to identify inhibitors of these enzymes, including those that inhibit all the three enzymes (manuscripts in preparation)
The ideal system to control half-life of CRISPR-associated nucleases and their technologies would have several characteristics. First, the system should be capable of degrading large protein:RNA complex (> 200 kDa); while SpCas9 has 1368 aa, adenine and cytosine base editors are made up of 1566 aa and 1835 aa, respectively. Base editors that can perform a simultaneous transformation of A→G and C→T are even larger, consisting of 2033 aa. Second, this system should be capable of effectively (~100 %) and rapidly (< 30 min) degrading these nucleases and their technologies to afford precision dose and temporal control as substantial editing is observed in as little as 30 min in cells. Here, a small-molecule control is preferable to other genetic methods-reduction techniques such as knockdown, which are slow to take effect. Third, the system should be minimalistic, consisting of just the controller (e.g., small molecule) and the engineered nuclease Cas9. Fourth, since CRISPR-based technologies (e.g., base editors) already bear N- and C-term fusions, genetic tags should be functional at an internal site (e.g., loops). We have developed chimeric small-molecules that can bring Cas9 in proximity to a ubiquitin ligase, enabling rapid ubiquitination and degradation of Cas9 by the proteasome. This system was able to control the Cas9 activity in a specific and dose-dependent manner, and its use ameliorated the off-target effects of the enzyme and biased the DNA repair pathways.