THE BROAD INSTITUTE, INC.;PRESIDENT AND FELLOWS OF HARVARD COLLEGE
发明人:
LIU, David R.,ANZALONE, Andrew Vito,NEWBY, Greg,EVERETTE, Kelcee
申请号：
USUS2020/023712
公开号：
WO2020/191233A1
申请日:
2020.03.19
申请国别(地区):
US
年份:
2020
代理人:
摘要：
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. 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 modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap, which is homologous to a strand of the targeted endogenous DNA sequence to be edited, but which contains the desired one or more nucleotide changes and which, following synthesis by the polymerase (e.g., reverse transcriptase), becomes incoporated into the target DNA molecule. Also disclosed herein are various methods that leverage prime editing, including treating trinucleotide repeat contraction diseases, installing targeted peptide tags, treating prion disease through the installation of protection mutations, manipulating RNA-encoding genes for the installation of RNA tags for controlling the function and expression of RNA, using prime editing to construct sophisticated gene libraries, using prime editing to insert immunoepitopes into proteins, use of prime editing to insert inducible dimerization domains into protein targets, and delivery methods, among others.La présente invention concerne des compositions et des procédés pour effectuer l'Édition primaire d'une molécule d'ADN cible (par exemple, un génome) qui permet l'incorporation d'un changement nucléotidique et/ou d'une mutagenèse ciblée. La modification nucléotidique peut compr
