Crispr oligoncleotides and gene editing

Abstract

The present disclosure generally relates to compositions and methods for the genetic modification of cells. In particular, the disclosure relates to CRISPR reagents and the use of such reagents.

1 . A method for producing a nucleic acid molecule, the method comprising performing polymerase chain reaction (PCR) in a reaction mixture containing (i) a double-stranded nucleic acid segment and (ii) at least one oligonucleotide capable of hybridizing to nucleic acid at one terminus of the double-stranded nucleic acid segment,
 wherein the nucleic acid molecule is produced by the PCR reaction, and   wherein the product nucleic acid molecule contains at or near one terminus a promoter suitable for in vitro transcription.   
     
     
         2 . The method of  claim 1 , wherein the nucleic acid molecule produced by the PCR reaction encodes an RNA molecule from 35 to 150 nucleotides in length. 
     
     
         3 . The method of  claim 1 , wherein the nucleic acid molecule produced by the PCR reaction is from 70 to 150 base pairs in length. 
     
     
         4 . The method of  claim 1 , wherein the nucleic acid molecule is produced by the PCR reaction encodes an RNA molecule with at least two hairpin turns. 
     
     
         5 . The method of  claim 1 , wherein the nucleic acid molecule is produced by the PCR reaction encodes a CRISPR RNA. 
     
     
         6 . The method of  claim 5 , wherein the nucleic acid molecule is produced by the PCR reaction encodes a guide RNA. 
     
     
         7 .- 26 . (canceled) 
     
     
         27 . A ligated RNA molecule comprising two RNA regions that differ in nucleotide sequence connected by a ligation group, wherein the ligated RNA molecule is capable of binding to a Cas9 protein and has a region of sequence complementarity of at least 10 nucleotides to a target locus. 
     
     
         28 . The ligated RNA molecule of  claim 27 , wherein one region is composed of a crRNA molecule and the other region is a tracrRNA molecule. 
     
     
         29 . A method of making the ligated RNA molecule of  claim 27 , the method comprising covalently linking a crRNA molecule and a tracrRNA molecule. 
     
     
         30 . A method for gene editing at a target locus within a cell, the method comprising forming an intracellular complex comprising a Cas9 protein and the ligated RNA molecule of  claim 27 , under conditions that allow for cleavage of the target locus. 
     
     
         31 . A method for performing homologous recombination in a cell, the method comprising:
 (a) introducing into the cell a nucleic acid cutting entity capable of generating a double-stranded break at a specified location in a nucleic acid molecule present inside a cell to produce a cleaved nucleic acid molecule, and   (b) introducing a donor nucleic acid molecule into the cell,   wherein step (a) is performed before step (b) or wherein step (b) is performed before step (a).   
     
     
         32 . The method of  claim 31 , wherein the introduction of the nucleic acid cutting entity or the donor nucleic acid molecule into the cell is mediated by electroporation. 
     
     
         33 . The method of  claim 31 , wherein the donor nucleic acid molecule is double-stranded and has either blunt termini or 5′ overhangs. 
     
     
         34 . The method of  claim 31 , wherein the donor nucleic acid molecule is single stranded. 
     
     
         35 . The method of  claim 31 , wherein the donor nucleic acid molecule contains one or more nuclease resistant group at one or both termini.