Dna polymerase-mediated genome editing
AssigneeWuhan University
InventorsHao YIN, Ying ZHANG, Shuhan LU, Jinlin WANG
Life of this patent
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Abstract
Provided are compositions and methods useful for inserting or replacing a nucleic acid fragment to a target genome sequence. Unlike the conventional prime editors, the disclosed methods do not require a retro-transcriptase or a pegRNA. Instead, the Cas protein is fused to, or otherwise coupled to, or is present within the same cell as, a DNA polymerase, which uses a single stranded donor DNA to generate the desired insertion sequence.
Claims21
A molecule comprising (a) a Cas protein and (b) a DNA polymerase, wherein the Cas protein is fused to the DNA polymerase or is coupled to the DNA polymerase through a covalent or ionic interaction, directly or indirectly.
A method for introducing a foreign nucleotide sequence into a target nucleotide, comprising contacting, in a cell, the target nucleotide with a Cas protein fused or coupled to (or co-present with in the cell) a DNA polymerase, a single guide RNA (sgRNA) comprising a spacer complementary to a protospacer in the target nucleotide, a donor single stranded DNA (ssDNA) that is complementary to a portion of the target nucleotide on the opposite strand of the protospacer, and further encodes the foreign nucleotide sequence.
A method for introducing a foreign nucleotide sequence into a target nucleotide, comprising contacting, in a cell, the target nucleotide with a Cas protein fused or coupled to a DNA polymerase,
- (a) a first single guide RNA (sgRNA) comprising a first spacer complementary to a first protospacer in the target nucleotide, a first donor single stranded DNA (ssDNA) that is complementary to a first portion of the target nucleotide on the opposite strand of the first protospacer, and further encodes a first portion of the foreign nucleotide sequence;
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- (b) a second single guide RNA (sgRNA) comprising a second spacer complementary to a second protospacer in the target nucleotide, a second donor single stranded DNA (ssDNA) that is complementary to a second portion of the target nucleotide on the opposite strand of the second protospacer, and further encodes the remaining portion of the foreign nucleotide sequence.
21-32. (canceled)
Dependencies are inferred from the claim text.