The present disclosure provides compositions and methods for primed editing of target
DNA molecules (e.g., genomes), which enable the incorporation of
nucleotide changes and / or targeted
mutagenesis.
Nucleotide changes can include single
nucleotide changes (e.g., any transition or any
transversion), the
insertion of one or more nucleotides, or the deletion of one or more nucleotides. More specifically, the present disclosure provides a
fusion protein comprising a
nucleic acid programmable
DNA binding protein (napDNAbp) and a
polymerase (e.g.,
reverse transcriptase), which is guided to a specific
DNA sequence by a modified
guide RNA termed PEgRNA. The PEgRNA is modified to include an extended portion (relative to the standard
guide RNA) that provides
a DNA synthesis template sequence. This encodes a single-stranded DNA flap that is homologous to the strand of the targeted endogenous DNA sequence to be edited, but which contains the desired one or more
nucleotide changes, and which becomes incorporated into the target DNA molecule after synthesis by the
polymerase (e.g.,
reverse transcriptase). Various methods utilizing prime editing are also disclosed herein, including, among others, treating trinucleotide repeat contraction diseases, incorporating targeted
peptide tags, treating prion diseases by incorporating protective mutations,
engineering genes encoding
RNA for the incorporation of
RNA tags to control
RNA function and expression, constructing high performance
gene libraries using prime editing, inserting immune epitopes onto proteins using prime editing, using prime editing to insert inducible dimerization domains onto
protein targets, and
delivery methods.