2025 Theses Doctoral

Combinatorial protein engineering to identify improved CRISPR activators

Giddins, Marla Jane

Laboratory-engineered proteins such as high-fidelity DNA polymerases, CRISPR base and primeeditors, and chimeric antigen receptors have transformed our ability to probe and manipulate biological systems. To craft these powerful tools, researchers fuse multiple domains into novel chimeras intended to retain the functional properties of their constituent parts. Although this approach has produced a number of important technologies, its low-throughout nature and high costs thwart efforts to explore complex combinatorial landscapes and limit our grasp on the “rules” governing synthetic protein assembly (e.g., which domains work best together, which domain orders are optimal, benefits of fusing multiple copies of the same domain, etc.). Previous state-of-the-art CRISPR activators, including the tripartite activator, VP64-P65- RTA (VPR) and the Synergistic Activation Mediator (SAM), have established the benefit of combining multiple activation domains (ADs) into a single complex for improved transcriptional modulation. While VPR and SAM have proven relatively successful in both in vitro and in vivo applications, neither activator shows uniform activity across targets and cell types. Furthermore, reports that these tools produce toxicity within cellular systems limit their utility in broad-ranging applications.

To probe a vast combinatorial landscape of multi-domain CRISPR activators while bypassing the arduous task of generating each construct one one-by-one, we developed a strategy for constructing large combinatorial libraries of protein variants en masse and used this method to functionally evaluate a library of >15,000 CRISPR activators. Importantly, we conduct our screen on multiple target genes to identify tools with consistent performance across the genome. Our findings bring to light a critical yet often overlooked feature of CRISPR activators: toxicity.

This work not only highlights the prevalence of this problem but also elucidates several biological factors that contribute to it. Our observation that many high-performing activators elicited minimal effects on cell fitness challenges the notion that toxicity is an inevitable byproduct of a potent activation – and suggests that this model greatly oversimplifies the nuanced relationship between these traits. We also explored how the biochemical properties of ADs (e.g., hydrophobicity and intrinsic disorder) and their combinatorial interactions drive activator performance. Finally, we identified two potent activators, MHV and MMH, that show enhanced activity across diverse targets and cell types over one of the gold-standard CRISPR activators, SAM. Our results underscore the power of high-throughput techniques for both improving our understanding of complex protein assemblies and identifying more powerful tools.