Versatile method for the detection of marker-free precision genome editing and genetic variation

A method for detecting a genetic modification in a DNA sequence of interest, comprising the steps of:

• (a) amplifying the DNA sequence of interest using a specially designed Type IIS restriction enzyme-tagging primer, comprising:
• (i) obtaining the DNA sequence of interest from a biological sample;
• (ii) synthesizing the Type IIS restriction enzyme-tagging primer based on the DNA sequence of interest;
• (iii) amplifying the DNA sequence of interest using the Type IIS restriction enzyme-tagging primer and a reverse primer;
• and
• (iv) purifying a Type IIS restriction enzyme-tagged amplicon;
• (b) digesting the Type IIS restriction enzyme-tagged amplicon with the Type IIS restriction enzyme;
• (c) isolating the smaller DNA fragment containing the genetic modification exposed in a 3′ single-stranded overhang;
• (d) capturing the genetic modification, comprising:
• (i) preparing a library of 16 DNA adaptors, wherein each DNA adaptor comprises one strand with sequence of 5′-CTGGGGCACGGGTAAGAAGCATTCTGTCTCTCTTCTAAGAATTCGAG CTCGGTACCCG-3′ (SEQ ID NO:
• 230);
• and one complementary strand with sequence of 5′-CGGGTACCGAGCTCGAATTCTTAGAAGAGAGACAGAATGCTTCTTAC CCGTGCCCCAGNN-3′ with “N” corresponding to A, T, G or C (SEQ ID NOs:
• 231-246), and wherein each DNA adaptor has a different “NN”;
• (ii) incubating the isolated smaller DNA fragment containing the 3′ overhang with the library of DNA adaptors and performing a ligation;
• and
• (iii) obtaining a ligated product;
• and
• (e) amplifying the ligated product to detect the presence of the genetic modification, wherein the DNA sequence of interest is a genomic locus or corresponds to a genomic locus of an RNA virus variant.