The present disclosure provides new prime editor guide RNAs for prime editing, constructs for prime editing, and methods for using same. In addition, the present disclosure provides compositions and methods for conducting prime editing of a target
DNA molecule (e.g., a
genome) that enables the incorporation of a
nucleotide change and / or targeted
mutagenesis (e.g.,
insertion or deletion). The
nucleotide change can include a single-
nucleotide change (e.g., any transition or any
transversion), an
insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides fusion proteins comprising
nucleic acid programmable
DNA binding proteins (napDNAbp) and a
polymerase (e.g.,
reverse transcriptase), which is guided to a specific
DNA sequence by a prime editor
RNA (PEgRNA). The prime editor
guide RNA comprises an extension arm that provides
a DNA synthesis template sequence which encodes a
single strand DNA flap, which is homologous to an endogenous DNA sequence, but which contains the desired one or more nucleotide changes and which, following synthesis by the
polymerase (e.g.,
reverse transcriptase), becomes incorporated into the target DNA molecule.