Modified nucleic acid editing systems for tethering donor dna

Abstract

The technology relates to a composition for tethering donor DNA to a nuclease, the composition comprising a nucleic acid comprising donor DNA and a consensus sequence for a DNA binding domain; and at least one of: a fusion protein comprising a nuclease coupled to a DNA binding domain for binding the consensus sequence; and a nucleic acid encoding the fusion protein.

1 . A composition for tethering donor DNA to a nuclease, the composition comprising a nucleic acid comprising donor DNA and a consensus sequence for a DNA binding domain; and at least one of:
 a fusion protein comprising a nuclease coupled to a DNA binding domain for binding the consensus sequence; and   a nucleic acid encoding the fusion protein.   
     
     
         2 . The composition of  claim 1  wherein the wherein the nuclease is a Cas protein, a Transcription activator-like effector nuclease (TALEN), a meganuclease, a Zinc Finger or a MADzyme. 
     
     
         3 . The composition of  claim 1  wherein the nuclease is a Cas protein. 
     
     
         4 . The composition of  claim 3  wherein the Cas protein is Cas9 
     
     
         5 . The composition of  claim 1  wherein the fusion protein further comprises a nuclear localization sequence. 
     
     
         6 . The composition of  claim 2 , further comprising a guide RNA that interacts with the Cas protein and a target DNA sequence. 
     
     
         7 . The composition of  claim 1  wherein the consensus sequence comprises the Lac operator (SEQ ID NO: 66), the TRP operator (SEQ ID NO: 68), the TET operator (SEQ ID NO 67), the GAL-4 binding site (SEQ ID NO: 1), or the IHF binding site (SEQ ID NO 2). 
     
     
         8 . The composition of  claim 7  wherein the consensus sequence comprises a sequence with at least 80%, 85%, 90%, 95% or at least 99% identity to the Lac operator, the TRP operator, the TET operator, the GAL-4 binding site, or the IHF binding site 
     
     
         9 . The composition of  claim 1  wherein the DNA biding domain comprises the LAC repressor, TET repressor, TRP-repressor, GAL-4, or IHF, or a portion thereof sufficient to bind the consensus sequence. 
     
     
         10 . The composition of  claim 1  wherein the DNA binding domain is the LAC repressor, preferably amino acids 43-403 of SEQ ID NO 9. 
     
     
         11 . The composition of  claim 1  wherein the nuclease is coupled to the DNA binding domain via a linker. 
     
     
         12 . The composition of  claim 11  wherein the linker comprises a sequence selected from any one of SEQ ID Nos: 3 to 7, a GGS linker, or amino acids 404-419 of SEQ ID NO 9. 
     
     
         13 . The composition of  claim 1  wherein the fusion protein comprises the LAC repressor and Cas9. 
     
     
         14 . The composition of  claim 1  comprising a vector, wherein the vector comprises either or both of:
 a. the nucleic acid comprising the donor DNA and the consensus sequence for a DNA binding domain; and 
 b. the nucleic acid encoding the fusion protein. 
 
     
     
         15 . The composition of  claim 14  wherein the vector further comprises a nucleic acid sequence encoding a guide RNA that interacts with the Cas protein and a target DNA sequence. 
     
     
         16 . The composition of  claim 1  wherein the fusion protein is nuclease deficient. 
     
     
         17 . An isolated host cell comprising the composition of  claim 1 . 
     
     
         18 . A method for editing DNA in a cell, the method comprising;
 contacting the cell with the composition of  claim 1  under conditions suitable for the interaction of the fusion protein with a target DNA sequence.   
     
     
         19 . A method for editing DNA in a cell, the method comprising
 a) contacting the cell with the composition of  claim 16  under conditions suitable for the interaction of the fusion protein with a first target DNA sequence; and   b) contacting the cell with a nucleic acid editing system adapted to edit the genomic DNA at a second target DNA sequence, under conditions suitable for nucleic acid editing.   
     
     
         20 . The method of  claim 18  wherein the target DNA sequence is selected from genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA. 
     
     
         21 . The method of  claim 18  wherein the efficiency of editing is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, or at least 99%. 
     
     
         22 . The method of  claim 18  wherein the cell is in a subject, preferably a human subject. 
     
     
         23 . A kit comprising:
 a nucleic acid comprising donor DNA and a consensus sequence for a DNA binding domain; and at least one of   a fusion protein comprising a nuclease coupled to a DNA binding domain for binding the consensus sequence; and   a nucleic acid encoding the fusion protein.   
     
     
         24 . The kit of  claim 23  wherein the nuclease is a Cas protein, a Transcription activator-like effector nuclease (TALEN), a meganuclease, a Zinc Finger or a MADzyme. 
     
     
         25 . The kit of  claim 24  wherein the Cas protein is Cas9.