Highly sensitive in vitro assays to define substrate preferences and sites of nucleic-acid binding, modifying, and cleaving agents
AssigneeThe General Hospital Corporation
InventorsJ. Keith Joung, Vikram Pattanayak, Karl Petri
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Abstract
Described herein are, among other things, methods of identifying double stranded DNA sequences that are modified by a base editing enzyme. For example, by providing a plurality of linear dsDNA oligonucleotides of known sequences, incubating the library in the presence of a base editing enzyme, amplifying the oliognucleotides with a polymerase that converts edited base pairs to equal mixtures of canonical base pairs during DNA synthesis, and determining the sequences of the amplified oligonucleotides.
Claims16
A method of identifying double stranded DNA sequences that are modified by a base editing enzyme, the method comprising:
- (i) providing a library of defined linear dsDNA oligonucleotides of known sequences, each oligonucleotide having a known sequence comprising, in order:
- a first sequence common to each of the oligonucleotides in the library;
- a first barcode sequence unique to the library member;
- a potential DNA substrate sequence for the base editing enzyme;
- a second copy of the barcode sequence;
- and
- a second sequence common to each of the oligonucleotides in the library;
- (ii) incubating the library in the presence of a base editing enzyme under conditions sufficient for modification to occur;
- (iii) amplifying the oligonucleotides with a polymerase that converts edited base pairs to equal mixtures of canonical base pairs during DNA synthesis, optionally wherein a dATP nucleotide is incorporated across from dU or a dCTP nucleotide is incorporated across from dI, such that an oligonucleotide that has been modified by the base editing enzyme will be amplified as a mixture of the original dsDNA oligonucleotide sequence from the library prior to incubation with the base editing enzyme and also a modified sequence that contains substitutions;
- and
- (iv) determining the sequences of the amplified oligonucleotides, thereby identifying double stranded DNA sequences that are modified by the base editing enzyme.
Dependencies are inferred from the claim text.