PROJECT SUMMARY CRISPR-Cas9 is an RNA-guided endonucleases that is being actively used for sequence-specific DNA recognition, genome engineering, targeted transcriptional activation/repression and genome imaging. Cas9 is being developed as a gene therapy agent for multiple pathologies, including HIV, vision disorders, muscular dystrophy, and hereditary disorders. The precision control of specificity of CRISPR-Cas9 is required as off- target effects and chromosomal translocations are observed at elevated activity. Further, the ease of targeting catalytically impaired Cas9 to any genomic locus has resulted in transformative technologies. For example, the fusion of catalytically inactive Cas9 (dCas9) to transcriptional activators or repressors has enabled gene transcription and repression; fusion of catalytically impaired Cas9 to base-modifying enzymes has allowed base conversion (e.g., C→T) at specific genomic sites; dCas9‒GFP fusion has made imaging genomic loci possible; and dCas9‒acetyltransferases or deacetylases fusion has enabled epigenome editing. We propose to apply chemical and genetic approaches develop reagents and methods that will allow precision control of specificity of CRISPR-Cas9. 1