CRISPR-Cas9 circular permutants as programmable scaffolds for genome modification

The ability to engineer proteins co-opted from nature to suit the needs of researchers is pivotal to a future, pragmatic biology. For example, CRISPR proteins adapted from bacterial immune systems are among the most effective tools used for genome modification and are revolutionizing biomedicine. Nuclease null CRISPR proteins can be fused to domains possessing alternative activities in order to rapidly activate, repress, or mutate the genome in a programmable fashion. Additionally, bacterial CRISPR immune systems are now being transplanted into new organisms to provide direct immunity or drive genes through a population with little control over their activity. These usages represent the future of CRISPR technology; yet there has been little research done to optimize the basic CRISPR-Cas9 scaffold to enable more efficient fusions or programmable activation of CRISPR tools by cellular effectors. Here, we demonstrate how the topological rearrangement of CRISPR-Cas9 using circular permutation can resolve many of these challenges. First, circular permutation of Cas9 can be used to position termini adjacent to bound DNA and offers a straightforward mechanism for improving many Cas9 fusion proteins. Circular permutation also revealed an unexpected mechanism by which engineered Cas9s can be caged until activation by pathogen-associated proteases. These protease-sensing Cas9s (ProCas9s) represent a unique class of single molecule effectors possessing both a designable input and programmable output. In proof of concept studies, we show that ProCas9s can sense a wide range of different proteases via an easily switchable input and devise a mechanism to enable an altruistic response upon sensing of a pathogen associated protease. Taken as a whole, this study provides a suite of novel CRISPR effectors specifically engineered to enable a safer and more efficient generation of genome modifying enzymes and molecular recorders for the advancement of precision genome engineering in research and biomedicine.