Dna editing using single-stranded dna

Abstract

Disclosed are compositions, methods, and kits for modifying DNA within cells as well as compositions and methods for modifying gene expression in a cell. In particular, the invention generally relates to compositions, methods, and kits for DNA editing using single-stranded DNA. Compositions and methods for modifying gene expression using artificial microRNAs (amiRNA) are also contemplated.

We claim: 
     
         1 . A method for modifying a target DNA sequence in a cell, the method comprising:
 (a) introducing a single-stranded DNA (ssDNA) in the cell, the ssDNA comprising a 5′ homology arm having substantial sequence identity to the target DNA sequence, an exogenous sequence, and a 3′ homology arm having substantial sequence identity to the target DNA sequence, wherein the ssDNA is between 200 and 10,000 nucleotides in length, and   (b) introducing or expressing a nuclease system in the cell, wherein the nuclease system cuts the target DNA sequence.   
     
     
         2 . The method of  claim 1 , wherein the ratio of the length of the exogenous sequence to the total length of the 5′ homology arm and the 3′ homology arm (exogenous sequence length:homology arm length) is between 1.5:1 and 20:1. 
     
     
         3 . The method of  claim 2 , wherein the exogenous sequence encodes a protein product, an RNA product, a DNA regulatory element, or a variant DNA sequence. 
     
     
         4 . The method of  claim 1 , wherein the ssDNA is produced by a method comprising:
 (a) transcribing a DNA template encoding a promoter operably linked to nucleotide sequence comprising a 5′ homology arm having substantial sequence identity to the target DNA sequence, an exogenous sequence, and a 3′ homology arm having substantial sequence identity to the target DNA sequence to produce a RNA transcript,   (b) synthesizing a ssDNA/RNA duplex by reverse transcription of the RNA transcript, and   (c) degrading the RNA from the ssDNA/RNA duplex using an RNA-degrading enzyme to produce ssDNA.   
     
     
         5 . The method of  claim 4 , further comprising purifying the ssDNA. 
     
     
         6 . The method of  claim 1 , wherein introducing a nuclease system in the cell comprises introducing into the cell a polynucleotide that encodes the nuclease system. 
     
     
         7 . The method of  claim 1 , wherein the nuclease system is selected from the group consisting of a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), an Argonaute nuclease system, and a CRISPR/Cas system. 
     
     
         8 . The method of  claim 7 , wherein the nuclease system comprises a CRISPR/Cas system. 
     
     
         9 . The method of  claim 8 , wherein the CRISPR/Cas system comprises a CRISPR/Cas9 system. 
     
     
         10 . The method of  claim 1 , wherein the cell is a prokaryotic cell. 
     
     
         11 . The method of  claim 1 , wherein the cell is a eukaryotic cell. 
     
     
         12 . The method of  claim 11 , wherein the cell is a mammalian cell. 
     
     
         13 . The method of  claim 12 , wherein the cell is a mouse cell. 
     
     
         14 . The method of  claim 1 , wherein the exogenous sequence encodes an artificial microRNA (amiRNA). 
     
     
         15 . A composition for modifying a target DNA sequence in a cell, the composition comprising:
 (a) single-stranded DNA (ssDNA), the ssDNA comprising a 5′ homology arm having substantial sequence identity to the target DNA sequence, an exogenous sequence, and a 3′ homology arm having substantial sequence identity to the target DNA sequence, wherein the ssDNA is between 200 and 10,000 nucleotides in length, and   (b) a nuclease system capable of cutting the target DNA sequence.   
     
     
         16 . A kit comprising an RNA polymerase, a reverse transcriptase, an RNA-degrading enzyme, and a nuclease system. 
     
     
         17 . The kit of  claim 16 , further comprising:
 single-stranded DNA (ssDNA), the ssDNA comprising a 5′ homology arm having substantial sequence identity to the target DNA sequence, an exogenous sequence, and a 3′ homology arm having substantial sequence identity to the target DNA sequence, wherein the ssDNA is between 200 and 10,000 nucleotides in length, and the nuclease system capable of cutting the target DNA sequence.