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CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

Author:
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.
Source:
Cell 2019 v.176 no.1-2 pp. 254-267.e16
ISSN:
0092-8674
Subject:
DNA, enzyme activity, gene editing, genome, medicine, proteinases, proteins, topology
Abstract:
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.
Agid:
6282795