Methods of use of CRISPR CPF1 hybrid DNA/RNA polynucleotides

Patent metadata

Field	Value
Publication number	US-11459588-B2
Application number	US-202117208146-A
Country	US
Kind code	B2
Filing date	Mar 22, 2021
Priority date	Jan 28, 2015
Publication date	Oct 4, 2022
Grant date	Oct 4, 2022

Abstract

The present disclosure provides DNA-guided CRISPR systems; polynucleotides comprising DNA, RNA and mixtures thereof for use with CRISPR systems; and methods of use involving such polynucleotides and DNA-guided CRISPR systems.

First claim

What is claimed is:

1. A method of modulating transcription of at least one gene within a target deoxyribonucleic acid (DNA) molecule, the method comprising:
contacting a target DNA molecule comprising a target sequence in vitro or in an isolated cell with
a single polynucleotide comprising a targeting region comprising a mixture of DNA and ribonucleic acid (RNA) and an activating region comprising a mixture of DNA and RNA, wherein the activating region is adjacent to the targeting region, the targeting region is configured to hybridize with the target sequence, and the activating region comprises a stem loop structure; and
a Cpf1 protein,

wherein the Cpf1 protein has no nuclease activity, and wherein the Cpf1 protein is capable of binding with the activating region of the single polynucleotide.

2. The method of claim 1 , wherein the target sequence is within an open reading frame sequence of the at least one gene.

3. The method of claim 1 , wherein the target sequence is within the promoter sequence of the at least one gene.

4. The method of claim 1 , wherein the contacting of the target DNA molecule with the single polynucleotide and the Cpf1 protein occurs in vitro.

5. The method of claim 1 , wherein the contacting of the target DNA molecule with the single polynucleotide and the Cpf1 protein occurs in an isolated cell.

6. The method of claim 5 , wherein the isolated cell is selected from the group consisting of a bacterial cell, an archaeal cell, a plant cell, an algal cell, a fungal cell, an invertebrate cell, a vertebrate cell, a mammalian cell, and a human cell.

7. The method of claim 1 , further comprising a polynucleotide encoding the Cpf1 protein.

8. The method of claim 1 , wherein the single polynucleotide and the Cpf1 protein form a complex.

9. The method of claim 1 , wherein the Cpf1 protein comprises a nuclear localization signal (NLS).

10. The method of claim 7 , wherein the single polynucleotide and the polynucleotide encoding the Cpf1 protein are introduced into the cell by lipofection, electroporation, nucleofection, microinjection, biolistics, liposomes, immunoliposomes, polycation, lipid:nucleic acid conjugates, or combinations thereof.

11. The method of claim 8 , wherein the single polynucleotide is introduced into the cell by lipofection, electroporation, nucleofection, microinjection, biolistics, liposomes, immunoliposomes, polycation, lipid:nucleic acid conjugates, or combinations thereof.

12. The method of claim 1 , wherein the polynucleotide comprises a compound selected from the group consisting of phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphorotriesters, aminoalkylphosphosphorotriesters, alkylphosphonates, 5′-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, 3′-amino phosphoramidate, amino alkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates.

Assignees

• Caribou Biosciences Inc

Inventors

• May Andrew Paul
• Donohoue Paul Daniel

Classifications

• C12N2320/51

  modulating the chemical stability, e.g. nuclease-resistance · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title

• C12N2310/10

  Type of nucleic acid · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title