Methods for variant detection

A method for detecting on-target and predicted off-target genome editing events, the method comprising:

• (a) providing a multiplex PCR reaction mixture comprising:
• (i) an on-target oligonucleotide primer having a cleavage domain position 5′ of a blocking group and a complementary region flanking the on-target genome edited locus, wherein the blocking group prevents primer extension and/or inhibits the oligonucleotide primer from serving as a template for DNA synthesis;
• (ii) one or more off-target oligonucleotide primers having a cleavage domain position 5′ of a blocking group and a complementary region flanking one or more predicted off-target genome edited loci, wherein the blocking group prevents primer extension and/or inhibits the oligonucleotide primer from serving as a template for DNA synthesis;
• (iii) a sample nucleic acid comprising a target DNA sequence, wherein the target DNA sequence has been altered with a gene editing enzyme;
• (iv) a cleaving enzyme;
• and
• (v) a polymerase, wherein the polymerase is a high-discrimination polymerase;
• (b) hybridizing the on-target oligonucleotide primer to the on-target genome edited locus to form an on-target double stranded substrate and hybridizing the one or more off-target oligonucleotide primers to the one or more predicted off-target genome edited loci to form an off-target double stranded substrate;
• (c) cleaving the on-target double stranded substrate with the cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group form the on-target oligonucleotide primer and cleaving the off-target double stranded substrate with the cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the off-target oligonucleotide;
• and
• (d) extending the on-target oligonucleotide primer and the off-target oligonucleotide primer with a polymerase.