A universal platform to enhance crispr-based gene editing for in vivo therapies

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.

We claim: 
     
         1 . A method for introducing a specific sequence into a target site on a target double-stranded nucleic acid in a cell, the method comprising
 (a) introducing into the cell or expressing within the cell a synthetic regulatory system comprising
 (i) a nucleotide sequence encoding a multifunctional Cas nuclease; 
 (ii) 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, whereby binding of the at least one truncated gRNA to the Cas nuclease directs the Cas nuclease to the nucleotide sequence encoding a NHEJ-associated enzyme; 
 (iii) at least one gRNA of 16 or greater nt in length that binds to or near the target site of the target double-stranded nucleic acid; and 
 (iv) a sequence encoding a donor nucleic acid molecule to be inserted into the target site; 
   
       wherein the nucleotide sequence encoding the Cas nuclease, the at least one truncated gRNA, and the at least one gRNA of 16 or greater nt comprise a single amplicon; and
 (b) inducing a double stranded break (DSB) at the target site, under conditions sufficient for sequence encoding the donor nucleic acid molecule to bind to the site of the DSB and the DSB to be repaired, whereby repair of the DSB introduces the sequence of the donor molecule into the target site. 
 
     
     
         2 . The method of  claim 1 , wherein the rate of homology-directed repair (HDR) compared with non-homologous end joining (NHEJ) is increased. 
     
     
         3 . The method of  claim 1 , wherein the multifunctional Cas nuclease is expressed as a fusion protein comprising a transcriptional activation or repression domain. 
     
     
         4 . The method of  claim 1 , wherein the multifunctional Cas nuclease is fused to a transcriptional repression domain and the truncated gRNA comprises an RNA aptamer for aptamer-mediated recruitment of the Cas nuclease. 
     
     
         5 . The method of  claim 4 , wherein the repression domain is KRAB-MecP2, MS2-KRAB-MecP2, or Com-KRAB. 
     
     
         6 . The method of  claim 1 , wherein the NHEJ-associated enzyme is selected from the group consisting of DNA ligase IV (LigIV), XRCC4, XRCC5 (KU80), and XRCC6 (KU70). 
     
     
         7 . The method of  claim 1 , wherein the system comprises four gRNAs of 15 or less nt in length, wherein each of the four gRNAs is complementary to at least a portion of a nucleotide sequence encoding a different NHEJ enzyme selected from the group consisting of DNA ligase IV (LigIV), XRCC4, XRCC5 (KU80), and XRCC6 (KU70). 
     
     
         8 . The method of  claim 1 , wherein the amplicon further comprises a truncated activating gRNA complementary to at least a portion of a nucleotide sequence encoding cell cycle progression factor, wherein the truncated activating gRNA further comprises a MS2 aptamer whereby binding of the truncated activating gRNA to the Cas nuclease directs the Cas nuclease to the nucleotide sequence for transcriptional activation. 
     
     
         9 . The method of  claim 8 , wherein the at least one cell cycle progression factor is selected from the group consisting of hepatocyte growth factor (HGF), Cyclin A1, Cyclin A2, Cyclin B1, Cyclin E1, skp2, CtIP, cyclin dependent kinase 2 (CDK2), and Geminin (GMNN). 
     
     
         10 . The method of  claim 8 , wherein the truncated activating gRNA further comprises a ligand-responsive riboswitch. 
     
     
         11 . The method of  claim 10 , wherein the ligand-responsive riboswitch is a tetracycline riboswitch or theophylline riboswitch. 
     
     
         12 . A method for efficient Homology-Directed repair (HDR)-based gene editing, the method comprising introducing into a cell a synthetic regulatory system comprising
 (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) enzyme;   (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;   (c) one or more RNA aptamers   
       wherein the at least one truncated gRNA and at least one gRNA, and the one or more aptamers comprise a single amplicon, and wherein the cell expresses a multifunctional Cas nuclease. 
     
     
         13 . The method of  claim 12 , wherein the amplicon further comprises a repression domain selected from the group consisting of KRAB-MecP2, MS2-KRAB-MecP2, and Com-KRAB.