Methods and compositions for identifying and enriching for cells comprising site specific genomic modifications

Abstract

The present invention relates to methods and compositions for modifying a target site in the genome of a plant cell. Such modifications include integration of a transgene and mutations. The present invention also relates to methods and compositions for identifying and enriching for cells which comprise a modified target site.

What is claimed is: 
     
         1 . A method of enriching for a cell comprising a transgene inserted into a nuclease cleavage site in a genome of the cell, comprising:
 a) introducing into a plurality of cells:   i) a first nucleic acid molecule comprising at least 100 contiguous nucleotides, wherein the at least 100 contiguous nucleotides have at least 90% identity with a target site in the genome of the cell, and further comprising a transgene; and   ii) a second nucleic acid molecule encoding a nuclease for site-directed cleavage at a nuclease cleavage site in the genome of the cell adjacent to the nucleotide sequence in the genome of the cell that corresponds to the at least 100 contiguous nucleotides of (i),   under conditions wherein expression of the second nucleic acid molecule can occur to produce the nuclease and the nuclease can cleave at the nuclease cleavage site in the genome of the cell and integrate the transgene into the nuclease cleavage site in the genome of the cell;   b) culturing the cells of (a) to produce at least one cell line or tissue;   c) extracting a genomic DNA sample from each of the cell lines or tissues of (b);   d) performing real-time quantitative polymerase chain reaction (qPCR) assays T and G on the samples of (c), wherein the assays T and G respectively comprise the following probes:   i) a first probe comprising a nucleotide sequence that is complementary to a nucleotide sequence of the target site, at least five base pairs away from the nuclease cleavage site for carrying out assay T, and   ii) a second probe comprising a nucleotide sequence that is complementary to a nucleotide sequence of the transgene for carrying out assay G;   e) obtaining a DNA copy number of the target site from the results of assay T and a DNA copy number of the transgene from the results of assay G; and   f) enriching for a cell line or tissue that has reduced copy number in assay T relative to a reference and a copy number greater than zero for assay G,   thereby enriching for the cell comprising the transgene inserted into the nuclease cleavage site in the genome of the cell.   
     
     
         2 . The method of  claim 1 , further comprising the step of discarding a cell line or tissue that has no change in the DNA copy number of assay T in comparison with a reference. 
     
     
         3 . The method of  claim 1 , further comprising the step of discarding a cell line or tissue that has a copy number of zero for assay G. 
     
     
         4 . The method of  claim 1 , wherein the assays are performed in the same mixture. 
     
     
         5 . The method of  claim 1 , wherein the cell line or tissue is derived from a plant or a plant part. 
     
     
         6 . The method of  claim 1 , wherein the plant is a maize plant. 
     
     
         7 . The method of  claim 1 , wherein the nuclease cleavage site is a maize MIR604 transgene insertion site within a nucleotide sequence, wherein said nucleotide sequence has at least 90% identity to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         8 . The method of  claim 1 , wherein the nuclease is an engineered nuclease with programmable cleavage target specificity. 
     
     
         9 . The method of  claim 8 , wherein the nuclease is a Cas9 nuclease. 
     
     
         10 . A method of identifying a cell comprising a transgene inserted into a nuclease cleavage site in a genome of the cell, comprising:
 a) introducing into a plurality of cells:   i) a first nucleic acid molecule comprising at least 100 contiguous nucleotides having at least 90% identity with a target site in the genome of the cell, and further comprising a transgene; and   ii) a second nucleic acid molecule encoding a nuclease for site-directed cleavage at a nuclease cleavage site in the genome of the cell adjacent to the nucleotide sequence in the genome corresponding to the at least 100 contiguous nucleotides of (i),   under conditions wherein expression of the second nucleic acid molecule can occur to produce the nuclease and the nuclease can cleave at the nuclease cleavage site in the genome of the cell and integrate the transgene into the nuclease cleavage site in the genome of the cell;   b) culturing the cells of (a) to produce at least one cell line or tissue;   c) extracting a genomic DNA sample from each of the cell lines or tissues of (b);   d) performing real-time quantitative polymerase chain reaction (qPCR) assays T and G on the samples of (c), wherein the assays T and G respectively comprise the following probes:   i) a first probe comprising a nucleotide sequence that is complementary to a nucleotide sequence of the target site, at least five base pairs away from the nuclease cleavage site for carrying out assay T, and   ii) a second probe comprising a nucleotide sequence that is complementary to a nucleotide sequence of the transgene for carrying out assay G;   e) obtaining a DNA copy number of the target site from the results of assay T and a DNA copy number of the transgene from the results of assay G; and   f) identifying a cell line or tissue that has reduced copy number in assay T relative to a reference and a copy number greater than zero for assay G,   thereby identifying a cell comprising the transgene inserted into the nuclease cleavage site in the genome of the cell.   
     
     
         11 . The method of  claim 10 , further comprising the step of discarding a cell line or tissue that has no change in the DNA copy number of assay T in comparison with a reference. 
     
     
         12 . The method of  claim 10 , further comprising the step of discarding a cell line or tissue that has a copy number of zero for assay G. 
     
     
         13 . The method of  claim 10 , wherein the assays are performed in the same mixture. 
     
     
         14 . The method of  claim 10 , wherein the cell line or tissue is derived from a plant or a plant part. 
     
     
         15 . The method of  claim 14 , wherein the plant is a maize plant. 
     
     
         16 . The method of  claim 10 , wherein the nuclease cleavage site is a maize MIR604 transgene insertion site within a nucleotide sequence, wherein said nucleotide sequence has at least 90% identity to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         17 . The method of  claim 10 , wherein the nuclease is an engineered nuclease with programmable cleavage target specificity. 
     
     
         18 . The method of  claim 17 , wherein the nuclease is a Cas9 nuclease. 
     
     
         19 . A cell line or tissue that is identified by the method of  claim 10 , wherein the cell line or tissue is from a plant. 
     
     
         20 . A plant or plant part, or progeny thereof, derived from the cell line or tissue of  claim 19 .