DNA-guided gene editing and regulation

Patent metadata

Field	Value
Publication number	US-11746349-B2
Application number	US-201716076022-A
Country	US
Kind code	B2
Filing date	Feb 7, 2017
Priority date	Feb 9, 2016
Publication date	Sep 5, 2023
Grant date	Sep 5, 2023

Abstract

Methods and compositions of altering a eukaryotic cell are described including providing to the eukaryotic cell a guide DNA sequence complementary to a target nucleic acid sequence, providing to the eukaryotic cell an Ago enzyme or a nuclease null Ago protein that interacts with the guide DNA sequence for DNA-guided gene editing and regulation of the target nucleic acid sequence in a site specific manner.

First claim

What is claimed is:

1. A method of altering a eukaryotic cell comprising
providing to the eukaryotic cell a guide DNA sequence complementary to a complementary target nucleic acid sequence,
providing to the eukaryotic cell an argonaute (Ago) enzyme that interacts with the guide DNA sequence and cleaves the complementary target nucleic acid sequence in a site specific manner, and
providing to the eukaryotic cell a nuclease null Cas9 protein and a guide RNA, wherein the guide RNA is complementary to a guide RNA targeting target nucleic acid sequence, wherein the nuclease null Cas9 and the guide RNA colocalize at the guide RNA targeting target nucleic acid sequence and unwind the guide RNA targeting target nucleic acid sequence,
wherein the guide DNA sequence binds to the complementary target nucleic acid sequence and the Ago enzyme cleaves the complementary target nucleic acid sequence in a site specific manner that is facilitated by the unwinding of the guide RNA targeting target nucleic acid sequence.

2. The method of claim 1 wherein the guide RNA targeting target nucleic acid sequence and the complementary target nucleic acid sequence are about 75 to 150 base pairs apart.

3. The method of claim 1 further including the step of providing a donor sequence and wherein the donor sequence is inserted into the complementary target nucleic acid sequence.

4. The method of claim 1 wherein the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.

5. The method or claim 1 wherein the eukaryotic cell is a human cell.

6. The method of claim 1 wherein a plurality of guide DNAs are provided into the cell that are complementary to different complementary target nucleic acid sequences and the Ago enzyme cleaves the different complementary target nucleic acid sequences in a site specific manner.

7. A method of altering a eukaryotic cell comprising
providing to the eukaryotic cell a first nucleic acid sequence that is complementary to a complementary target nucleic acid sequence,
providing to the eukaryotic cell a second nucleic acid sequence encoding an argonaute (Ago) enzyme that interacts with the first nucleic acid sequence and cleaves the complementary target nucleic acid sequence in a site specific manner, and
providing to the eukaryotic cell a nuclease null Cas9 protein and a guide RNA, wherein the guide RNA is complementary to a guide RNA targeting target nucleic acid sequence,
wherein the nuclease null Cas9 and the guide RNA colocalize at the guide RNA targeting target nucleic acid sequence and unwind the guide RNA targeting target nucleic acid sequence,
wherein the first nucleic acid sequence is a guide DNA, wherein the second nucleic acid sequence is expressed, and
wherein the guide DNA sequence binds to the complementary target nucleic acid sequence and the Ago enzyme that is expressed from the second nucleic acid sequence cleaves the complementary target nucleic acid sequence in a site specific manner that is facilitated by the unwinding of the guide RNA targeting target nucleic acid sequence.

8. The method of claim 7 further including the step of providing a donor sequence and wherein the donor sequence is inserted into the complementary target nucleic acid sequence.

9. The method of claim 7 wherein the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.

10. The method or claim 7 wherein the eukaryotic cell is a human cell.

11. The method of claim 7 wherein a plurality of guide DNAs are provided into the cell that are complementary to different complementary target nucleic acid sequences and the Ago enzyme cleaves the different complementary target nucleic acid sequences in a site specific manner.

12. The method of claim 7 wherein the second nucleic acid sequence is present on a recombinant expression vector.

13. The method of claim 7 wherein the guide RNA targeting target nucleic acid sequence and the complementary target nucleic acid sequence are about 75 to 150 base pairs apart.

14. An in vitro eukaryotic cell comprising
a first nucleic acid sequence that is complementary to a complementary target nucleic acid sequence,
a second nucleic acid sequence encoding an argonaute (Ago) enzyme that interacts with the first nucleic acid sequence and cleaves the complementary target nucleic acid sequence in a site specific manner,
a nuclease null Cas9 protein and a guide RNA, wherein the guide RNA is complementary to a guide RNA targeting target nucleic acid sequence, wherein the nuclease null Cas9 and the guide RNA colocalize at the guide RNA targeting target nucleic acid sequence and unwind the guide RNA targeting target nucleic acid sequence,
wherein the first nucleic acid sequence is a guide DNA, and wherein the cell expresses the Ago enzyme, and wherein the guide DNA binds to the complementary target nucleic acid and the Ago enzyme cleaves the complementary target nucleic acid sequence in a site specific manner that is facilitated by the unwinding of the guide RNA targeting target nucleic acid sequence.

15. The eukaryotic cell of claim 14 wherein the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.

16. The eukaryotic cell of claim 14 wherein the eukaryotic cell is a human cell.

17. The eukaryotic cell of claim 14 wherein the cell further includes a plurality of nucleic acids encoding a plurality of guide DNA sequences complementary to different complementary target nucleic acid sequences.

18. The eukaryotic cell of claim 14 wherein the guide RNA targeting target nucleic acid sequence and the complementary target nucleic acid sequence are about 75 to 150 base pairs apart.

Assignees

• Harvard College

Inventors

• Church George M
• Yang Luhan
• Monroe Margo R
• Huang Po-Yi

Classifications

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• C12Y306/04013

  RNA helicase (3.6.4.13) · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title