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Open Access Publications from the University of California

CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification.

  • Author(s): 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
  • et al.

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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.

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This item is under embargo until December 24, 2019.

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