CRISPR Oligonucleotides 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 is 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 is 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 method for gene editing at a target locus in a cell, the method comprising:
 (A) introducing into the cell a CRISPR protein and a CRISPR RNA molecule, wherein the CRISPR RNA has a region of sequence complementarity of at least 10 base pairs to the target locus, and   (B) introducing into the cell a donor DNA molecule,   wherein the CRISPR protein and the CRISPR RNA molecule is introduced into the cell first before the DNA molecule is introduced into the cell, and   wherein the CRISPR RNA molecules has two regions of sequence complementarity with a target locus in the cell.   
     
     
         28 . The method of  claim 27 , wherein the CRISPR protein and the CRISPR RNA molecules comprise a Cas9/gRNA complex. 
     
     
         29 . The method of  claim 27 , wherein the CRISPR protein and the CRISPR RNA molecules are introduced into the cell by electroporation. 
     
     
         30 . The method of  claim 27 , wherein the donor DNA molecule is single-stranded. 
     
     
         31 . The method of  claim 27 , wherein the donor DNA molecule is has two regions of sequence complementarity to the target locus in the cell and an intervening region. 
     
     
         32 . The method of  claim 31 , wherein the donor DNA molecule comprises two regions of sequence complementarity to the target locus in the cell that are independently between 30 and 50 nucleotides in length. 
     
     
         33 . A method for introducing different CRISPR RNA molecules into cells, the method comprising contacting different samples of the cells with different CRISPR RNA molecules,
 wherein the different CRISPR RNA molecules are combined with a CRISPR protein prior to contacting the different samples of the cells, and   wherein, prior to introduction in the cells, the CRISPR protein is stored under conditions where the CRISPR protein will retain at least 75% activity after six months at 4° C.   
     
     
         34 . The method of  claim 33 , wherein the CRISPR protein is stored with a transfection reagent, a cell culture medium, or a CRISPR RNA molecule. 
     
     
         35 . The method of  claim 33 , wherein the CRISPR protein is stored in separate locations. 
     
     
         36 . The method of  claim 35 , wherein the separate locations are wells of a multi-well plate. 
     
     
         37 . The method of  claim 35 , wherein the different CRISPR RNA molecules are individually added to the separate locations where the CRISPR protein is stored.