Patent ID: 11898203
Assignee: THE GENERAL HOSPITAL CORPORATION
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 7:
8. An in vitro method of identifying double stranded DNA sequences that are modified by an adenine base editing enzyme that converts deoxyadenine to deoxyinosine and generates a nick on the opposite strand, the method comprising:
(i) providing a library of defined linear dsDNA oligonucleotides of known sequences, each library member having a first strand comprising, from 5′ to 3′:
a first known common sequence common to each of the oligonucleotides in the library;
a first known barcode sequence unique to the library member;
one and only one copy of a known potential DNA substrate sequence for the adenine base editing enzyme comprising a cognate protospacer adjacent motif (PAM);
a second known barcode sequence identical to the first barcode sequence; and
a second known common sequence common to each of the oligonucleotides in the library; and
a second strand complementary to the known sequences of the first strand;
(ii) incubating the library of linear dsDNA oligonucleotides in the presence of the adenine base editing enzyme under conditions sufficient for conversion of deoxyadenine to deoxyinosine to occur on one strand and nick generation to occur on the opposite strand of one or more of the linear dsDNA oligonucleotides and then incubating the modified linear dsDNA oligonucleotide(s) in the presence of endonuclease V to generate a single-strand break at sites with inosine nucleotides, creating a staggered double-strand break in the modified linear dsDNA oligonucleotide(s), and thereby creating first and second dsDNA oligonucleotide fragments with 5′ phosphorylated overhangs;
(iii) incubating the dsDNA oligonucleotide fragments with a DNA polymerase that creates 5′ phosphorylated blunt ends from the 5′ phosphorylated overhangs;
(iv) ligating the 5′ phosphorylated blunt ends with double stranded DNA adapters comprising primer sequences;
(v) amplifying the first dsDNA oligonucleotide fragments using one primer specific to the primer sequence of the DNA adapter and one primer specific to the first common sequence and amplifying the second dsDNA oligonucleotide fragments using one primer specific to the primer sequence of the DNA adapter and one primer specific to the second common sequence;
and
(vi) determining the sequence of a barcode sequence of one or more of the amplified dsDNA oligonucleotide fragments thereby identifying double stranded DNA sequences that are modified by the base editing enzyme.