METHODS AND COMPOSITIONS FOR EDITING NUCLEOTIDE SEQUENCES
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.
1 - 151 . (canceled)
152 . A gene editing composition comprising:
a) a protein complex comprising:
a) a zinc finger nuclease with an active nuclease domain; and
b) a polymerase;
b) a zinc finger nuclease with an inactive nuclease domain; and
c) an extension arm comprising a DNA synthesis template and a primer binding site.
153 . The gene editing composition of claim 152 , wherein the extension arm is chemically coupled to the zinc finger nuclease with an inactive nuclease domain.
154 . The gene editing composition of claim 152 , wherein the active nuclease domain is a Fok1 nuclease domain.
155 . The gene editing composition of claim 152 , wherein the inactive nuclease domain is a Fok1 nuclease domain.
156 . The gene editing composition of claim 152 , wherein the polymerase is attached to the zinc finger nuclease via a chemical linker.
157 . The gene editing composition of claim 152 , wherein the polymerase is attached to the zinc finger nuclease via an amino acid linker.
158 . The gene editing composition of claim 152 , wherein the polymerase is a DNA-dependent DNA polymerase.
159 . The gene editing composition of claim 152 , wherein the polymerase is an RNA-dependent DNA polymerase.
160 . The gene editing composition of claim 152 further comprising a helicase.
161 . The gene editing composition of claim 152 further comprising a FEN1.
162 . A gene editing composition comprising:
a) a protein complex comprising:
a) a transcription activator-like effector nuclease (TALEN) with an active nuclease domain; and
b) a polymerase;
b) a TALEN with an inactive nuclease domain; and
c) an extension arm comprising a DNA synthesis template and a primer binding site.
163 . The gene editing composition of claim 162 , wherein the extension arm is chemically coupled to the TALEN with an inactive nuclease domain.
164 . The gene editing composition of claim 162 , wherein the active nuclease domain is a Fok1 nuclease domain.
165 . The gene editing composition of claim 162 , wherein the inactive nuclease domain is a Fok1 nuclease domain.
166 . The gene editing composition of claim 162 , wherein the polymerase is attached to the TALEN via a chemical linker.
167 . The gene editing composition of claim 162 , wherein the polymerase is attached to the TALEN via an amino acid linker.
168 . The gene editing composition of claim 162 , wherein the polymerase is a DNA-dependent DNA polymerase.
169 . The gene editing composition of claim 162 , wherein the polymerase is an RNA-dependent DNA polymerase.
170 . The gene editing composition of claim 162 further comprising a helicase.
171 . The gene editing composition of claim 162 further comprising a FEN1.