The present invention provides a Cas9 platform to facilitate single-site
nuclease gene editing precision within a human
genome. For example, a Cas9
nuclease /
DNA-targeting unit (Cas9-DTU)
fusion protein precisely delivers a Cas9 / sgRNA complex to a specific
target site within the
genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9
target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated
DNA cleavage fidelity. Cas9-DTU fusion proteins have improved
target site binding precision, greater
nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9
genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.