Ornelas, I. J.; Owens, L. A.; Alamos, S.; Hummel, N. F.; Hernandez Zamora, M. G.; Phan, N. T.; Pattali, R. K.; Shih, P. M.; Nunez, J. K.

Chromatin is essential for eukaryotic life. Tens of thousands of chromatin regulator (CR) proteins exist in eukaryotic genomes that are predicted to modulate chromatin states; however, their molecular functions remain largely untested experimentally. Here, we construct a library of over 300 full-length CRs from humans, plants, yeast, protozoa, and virus, each fused to DNA-binding domains, and test their direct effect on transcriptional repression and activation in plants and human cells. We discover CRs with cross-kingdom functionality when transferred across eukaryotes, including CRs that outperform existing tools for programmable transcriptional repression and activation in plants and human cells. Using pooled CRISPR screens, we demonstrate a suite of CRISPR repressors that titrate gene expression at intermediate levels. Finally, we identify RCOR1 and MTA2 as universal eukaryotic repressors that retain repressive activity in plants, yeast, and human cells. Our toolkit advances synthetic eukaryotic engineering and expands our understanding of CR functionality across eukaryotes.