Methods of identifying and characterizing gene editing variations in nucleic acids

Abstract

Compositions and methods of identifying and characterizing potential gene editing on-target and off-target sites and/or edits in a nucleic acid are provided.

1 . A method of identifying and characterizing variations of an edited polynucleotide, comprising:
 (a) creating at least one edit in a polynucleotide at an intended target site,   (b) capturing the polynucleotide that comprises the intended target site, an off-target site, or a combination thereof,   (c) amplifying the captured polynucleotide of (b) to create a pool of polynucleotides,   (d) sequencing the pool of (c), and   (e) assessing the pool of polynucleotides to identify sequences corresponding to the intended target site, sequences corresponding to an off-target site, or a combination thereof.   
     
     
         2 . The method of  claim 1 , further comprising generating nucleic acid fragments of the captured polynucleotide and recovering said fragments to create an enriched DNA pool. 
     
     
         3 . The method of  claim 2 , further comprising characterizing the sequence composition of the enriched DNA pool to determine the nature of the enriched pool. 
     
     
         4 . The method of  claim 3 , wherein the nature of the enriched pool comprises the composition and abundance of each sequenced fragment species. 
     
     
         5 .- 13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the polynucleotide in step (b) is captured by a molecule that binds to said polynucleotide but lacks substantial nuclease activity. 
     
     
         15 . The method of  claim 14 , wherein said molecule is a deactivated Cas9 (dCas9), wherein the dCas9 is complexed with a guide polynucleotide corresponding to a target sequence of interest on the polynucleotide of step (a). 
     
     
         16 . The method of  claim 14 , wherein said molecule further comprises a tag for protein purification or isolation. 
     
     
         17 . The method of  claim 14 , wherein said tag is selected from the group consisting of: His tag, FLAG tag, HA tag, chitin binding protein (CBP) tag, maltose binding protein (MBP) tag, glutathione-S-transferase (GST) tag, thioredoxin (TRX) tag, poly(NANP) tag, V5-tag, Myc-tag, and NE-tag. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 15 , further comprising eluting the guide polynucleotide-dCas9 protein-polynucleotide complex created in step (b), and wherein step (e) is replaced with comparing the sequence of the target polynucleotide to a reference nucleic acid sequence to determine whether the guide polynucleotide directed the dCas9 to bind an intended target site on the polynucleotide of (a), a potential off-target site, or combination thereof. 
     
     
         20 .- 32 . (canceled) 
     
     
         33 . The method of  claim 1 , wherein the polynucleotide in step (b) is captured by a labeled probe. 
     
     
         34 . The method of  claim 33 , further comprising shearing of the polynucleotide prior to the capture step of (b). 
     
     
         35 . The method of  claim 1 , further comprising comparing at least one sequence of (e) to a reference nucleic acid sequence. 
     
     
         36 . The method of  claim 35 , comprising aligning said at least one sequence of (e) with the reference nucleic acid sequence and identifying at least one difference between said sequence and the reference nucleic acid sequence. 
     
     
         37 . The method of  claim 35 , wherein the reference sequence does not comprise said target site edit, said off-target site edit, or combinations thereof. 
     
     
         38 . The method of  claim 1 , further comprising cutting the captured polynucleotide of (b) into smaller fragments using random shearing or restriction digestion to generate a target site library of fragments. 
     
     
         39 . The method of  claim 1 , wherein the polynucleotide of (a) is in an in vitro environment. 
     
     
         40 . The method of  claim 1 , wherein the polynucleotide of (a) is from an oligonucleotide target site library. 
     
     
         41 . The method of  claim 40 , wherein the target site library is a randomer nucleotide combinatorial target site library. 
     
     
         42 .- 49 . (canceled) 
     
     
         50 . The method of  claim 1 , comprising performing steps (b), (c), and (d) in parallel so that multiple nucleic acids can be characterized. 
     
     
         51 . (canceled) 
     
     
         52 . The method of  claim 1 , further comprising additional editing of the polynucleotide based on the assessment of the presence or absence of the intended target site edit, off-target site edit, or combination thereof. 
     
     
         53 . The method of  claim 1 , the method further comprising: evaluating the genotype or phenotype of a cell or an organism comprising the intended target site edit, off-target site edit, or combinations thereof, optionally under various conditions, at one or more time point(s), in one or more cell type(s) or tissue(s), or in different environments. 
     
     
         54 . (canceled) 
     
     
         55 . (canceled) 
     
     
         56 . The method of claim  , further comprising selecting at least one cell or individual of said organism that comprises said intended target site. 
     
     
         57 . The method of  claim 53 , further comprising selecting at least one cell or individual of said organism that does not comprise at least one off-target site. 
     
     
         58 . The method of  claim 56 , further comprising growing said cell or organism. 
     
     
         59 . The method of  claim 56 , further comprising reproducing said cell or organism. 
     
     
         60 . The method of  claim 56 , further comprising crossing said organism with another to obtain a progeny, and evaluating said progeny for the presence or absence of the target and/or off-target sites. 
     
     
         61 . The method of  claim 58 , further comprising selecting a progeny based on the determination of the presence or absence of the determined off-target site edit, the intended target site edit, or combinations thereof. 
     
     
         62 . The progeny of the organism of  claim 58 , wherein said progeny comprises the intended target site edit in its genome. 
     
     
         63 . The progeny of the organism of  claim 58 , wherein said progeny does not comprise at least one off-target site edit in its genome. 
     
     
         64 . The method of  claim 60 , further comprising additional editing of the polynucleotide based on the assessment of the presence or absence of the intended target site edit, off-target site edit, or combination thereof in said organism or progeny. 
     
     
         65 . The method of  claim 53 , wherein said organism is a plant, mammal, virus, insect, fungus, or microorganism. 
     
     
         66 . The method of  claim 65 , wherein said plant is selected from the group consisting of: maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, switchgrass, soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tobacco, Arabidopsis, vegetable, and safflower. 
     
     
         67 .- 72 . (canceled) 
     
     
         73 . A method for generating a portfolio of intended target sites, potential off-target sites or combinations thereof within a genome of interest, comprising:
 (a) creating at least one nucleotide variation at an intended target site in a polynucleotide,   (b) capturing said polynucleotide that comprises at least one intended target site, an off-target site, or a combination thereof,   (c) amplifying the captured polynucleotide of (b) to create a pool of polynucleotides,   (d) sequencing the pool of (c),   (e) identifying from the pool of polynucleotides to sequences corresponding to the intended target site, sequences corresponding to an off-target site, or a combination thereof, and   (f) selecting at least one sequence from (e) to include in the portfolio.   
     
     
         74 . The method of  claim 73 , wherein the sequence of (f) is represented in silico. 
     
     
         75 . (canceled) 
     
     
         76 . The method of  claim 73 , wherein the sequence of (f) is stored as a polynucleotide in a cell. 
     
     
         77 . The method of  claim 73 , comprising a plurality of sequences of (f). 
     
     
         78 . A nucleic acid target portfolio comprising a library of intended target sites and/or potential off-target sites generated from the method of  claim 73 .