Highly sensitive in vitro assays to define substrate preferences and sites of nucleic-acid binding, modifying, and cleaving agents

A method of identifying double stranded DNA sequences that are modified by a base editing enzyme, the method comprising:

• (i) providing a plurality of linear dsDNA oligonucleotides of known sequences, each oligonucleotide having a 5′ end and a 3′ end and bearing two copies of a unique identifier sequence at or near both the 3′ and 5′ ends of the oligonucleotide, and a common sequence that is present in each one of the oligonucleotides in the plurality;
• (ii) incubating the plurality linear dsDNA oligonucleotides 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 barcode-linked sequence from the pre-treatment library 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.