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.
Published Web Locationhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414052/
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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