High-throughput genome engineering

National Human Genome Research Institute

Investigators

Linked publications, trials & patents

Abstract

The project produced five publications in the last reporting period. Three of these papers were fully from the SBGE section. We performed deep mutational scanning of a human innate immune protein, PKR, to determine its resilience to mutagenesis and its propensity to escape inhibition by a viral inhibitor (ref 1). Our work helped to explain how some proteins can gain beneficial mutations while maintaining their core function. In another paper, we developed a method to generate all possible in-frame deletions in a gene of interest, and applied it to the anti-CRISPR protein AcrIIA4 (ref 2). Our work revealed that AcrIIA4 requires its contacts with the PAM-binding domain of Cas9, but not its contacts with the catalytic domain. For this paper, we developed a method that can be widely used to identify the functionally important parts of a protein. In the third paper, we described our idea that high-throughput pooled experiments - typically used to test thousands of hypotheses at once - also have huge potential to help in "everyday" experiments testing one or a few focused hypotheses (ref 3). This work could have enormous impact on the reproducibility of scientific experiments. The last two publications were collaborative efforts. In collaboration with Jeff Lewis's lab at the University of Arkansas, we generated a set of vectors for editing by the CRISPEY system in diverse yeast strains (ref 4). These vectors will make it easier to expand the kinds of studies done in laboratory yeast to diverse yeast strains. And in collaboration with Chris MacDonald's lab at the University of York, we dissected why yeast strains with deletions in COG complex genes are sensitive to the toxin K28 (ref 5). This study showed the importance of proper subcellular localization of defense factors against toxins.