Discovery of hypercompact epigenetic modulators for persistent CRISPR-mediated gene activation

Programmable epigenetic modulators provide a powerful toolkit for controlling gene expression in novel therapeutic applications, but recent discovery efforts have primarily selected for potency of effect rather than contextual robustness or durability thereof. Current CRISPR-based tools are further limited by large cargo sizes that impede clinical delivery and, in gene activation contexts, by brief activity windows that preclude transient, single-dose strategies such as lipid nanoparticle (LNP) delivery. To address these limitations, we perform high-throughput screening to discover novel classes of transcriptional modulators derived from thousands of human, viral, and archaeal proteomes. We identify high-potency activators capable of mitotically stable gene activation in a multitude of cellular contexts and leverage machine learning models to rationally engineer variants with improved activities. In liver and T-cells, novel hypercompact activators (64 to 98 amino acids) derived from vIRF2 core domain (vCD) achieve superior potency and durable activation lasting weeks beyond the current large activators (~five-fold larger). In a humanized mouse model, we target a human hypercholesterolemia susceptibility gene and achieve activation persisting five weeks after a single dose by LNP delivery. Our discovery pipeline provides a predictive rubric for the development of contextually robust, potent, and persistent activators of compact size, broadly advancing the therapeutic potential of epigenetic gene activation. ### Competing Interest Statement L.S.Q. is founder and shareholder of Epicrispr Biotechnologies, and scientific advisor of Laboratory of Genomic Research and Kytopen. G.A.C., R.W.Y., T.B.G., M.Z.J., X.Y., V.C., J.K., K.J., N.J.S, S.B., A.C.D., L.S.Q., T.P.D., and D.O.H. are inventors on provisional patents relating to this work, and/or are employees of and acknowledge outside interest in Epicrispr Biotechnologies.