Universal platform to enhance CRISPR-based gene editing for in vivo therapies
AssigneeArizona Board of Regents on behalf of Arizona State University
InventorsSamira Kiani, Mo Reza Ebrahimkhani, Jennifer Chapman
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Abstract
Aspects of the disclosure relate to methods and synthetic regulatory systems for more efficient nuclease-mediated homology-directed repair (HDR). In particular, provided herein are methods for more efficient in vivo and in vitro HDR-based gene editing where the methods comprise introducing into a cell a synthetic regulatory system comprising Cas nuclease, guide RNAs (gRNAs) having various lengths and configured to target distinct nucleotide sequences for simultaneous transcriptional repression (or activation) and genome editing via double stranded break and use of a donor nucleic acid molecule as a template for repair.
Claims8
A method for efficient Homology-Directed repair (HDR)-based gene editing, the method comprising introducing into an isolated recombinant host cell:
- (a) at least one truncated guide RNA (gRNA) of 15 or less nucleotides (nt) in length complementary to at least a portion of a nucleotide sequence encoding a non-homologous end joining (NHEJ)-associated enzyme selected from the group consisting of DNA ligase IV (LigIV), XRCC4, XRCC5 (KU80), and XRCC6 (KU70);
- (b) at least one gRNA of 16 or greater nt in length that is complementary to at least a portion of a gene targeted for genetic editing;
- and
- (c) an RNA aptamer that recruits methyl-CpG binding protein (MecP2);
- wherein the at least one truncated gRNA, the at least one gRNA, and the aptamer comprise a single amplicon;
- and
- wherein the host cell expresses a catalytically active Cas9 nuclease.
Dependencies are inferred from the claim text.