Journal
CELL
Volume 176, Issue 1-2, Pages 254-+Publisher
CELL PRESS
DOI: 10.1016/j.cell.2018.11.052
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Funding
- Innovative Genomics Institute entrepreneurial fellowship program
- U.S. NIH K99/R00 Pathway to Independence Award from the NIGMS [K99GM118909, R00GM118909]
- HHMI
- U.S. NIH New Innovator Award from the NIBIB [1DP2EB018658-01]
- NIH S10 instrumentation grants [S10RR029668, S10RR027303]
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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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