公开(公告)号：US20210395710A1

公开(公告)日：2021-12-23

申请号：US17313328

申请日：2021-05-06

Applicant: University of Massachusetts

Inventor： Scot A Wolfe ,  Mehmet Fatih Bolukbasi ,  Ankit Gupta ,  Erik J. Sontheimer ,  Nadia Amrani

IPC: C12N9/22

Abstract: 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.