Main content area

CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

Oakes, Benjamin L., Fellmann, Christof, Rishi, Harneet, Taylor, Kian L., Ren, Shawn M., Nadler, Dana C., Yokoo, Rayka, Arkin, Adam P., Doudna, Jennifer A., Savage, David F.
Cell 2019 v.176 no.1-2 pp. 254-267.e16
DNA, enzyme activity, gene editing, genome, medicine, proteinases, proteins, topology
The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.