Systems, methods, and compositions for targeted nucleic acid editing

Patent metadata

Field	Value
Publication number	US-11866697-B2
Application number	US-201816614549-A
Country	US
Kind code	B2
Filing date	May 18, 2018
Priority date	May 18, 2017
Publication date	Jan 9, 2024
Grant date	Jan 9, 2024

Abstract

The invention provides for systems, methods, and compositions for targeting and editing nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA-targeting systems comprising a DNA-targeting Cpf1 protein, at least one guide molecule, and at least one adenosine deaminase protein or catalytic domain thereof.

First claim

What is claimed is:

1. A method of modifying an Adenine in a target locus of interest, comprising delivering to said locus:
(a) a Cpf1 nickase protein;
(b) a guide molecule which comprises a guide sequence linked to a direct repeat sequence; and
(c) an adenosine deaminase protein or catalytic domain thereof;
wherein said adenosine deaminase protein or catalytic domain thereof is covalently or non-covalently linked, or is adapted to covalently or non-covalently link after delivery, to said Cpf1 nickase protein or said guide molecule;
wherein said guide molecule forms a complex with said Cpf1 nickase protein and directs said complex to bind a first DNA strand at said target locus of interest, wherein said guide sequence is capable of hybridizing with a target sequence comprising said Adenine within said first DNA strand to form a heteroduplex, wherein said guide sequence comprises a non-pairing Cytosine at a position corresponding to said Adenine resulting in an A-C mismatch in the heteroduplex formed;
wherein said Cpf1 nickase protein nicks a second DNA strand at said target locus of interest displaced by formation of said heteroduplex; and
wherein said adenosine deaminase protein or catalytic domain thereof deaminates said Adenine in said heteroduplex.

2. The method of claim 1 , wherein said adenosine deaminase protein or catalytic domain thereof
is fused to the C-terminus of said Cpf1 nickase protein; or
is linked to an adaptor protein and said guide molecule or said Cpf1 nickase protein comprises an aptamer sequence capable of binding to said adaptor protein.

3. The method of claim 2 , wherein said adenosine deaminase protein or catalytic domain thereof is fused to said Cpf1 nickase protein by a linker.

4. The method of claim 3 , wherein said linker is (GGGGS) 3-11 (SEQ ID NOS:1-9), GSG5 (SEQ ID NO:10) or LEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO:11).

5. The method of claim 2 , wherein said adaptor sequence is selected from MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1.

6. The method of claim 1 , wherein said Cpf1 nickase protein comprises a mutation in the Nuc domain;
comprises a mutation corresponding to R1226A in AsCpf1; or
has at least part of the Nuc domain removed.

7. The method of claim 1 , wherein said guide molecule binds to said Cpf1 nickase protein and
is capable of forming said heteroduplex of about 24 nt with said target sequence; or
is capable of forming said heteroduplex of more than 24 nt with said target sequence.

8. The method of claim 1 , wherein said adenosine deaminase protein or catalytic domain thereof
is a human, squid or Drosophila adenosine deaminase protein or catalytic domain thereof;
has been modified to increase activity against a DNA-RNA heteroduplex;
is a mutated hADAR2d comprising mutation E488Q or a mutated hADAR1d comprising mutation E1008Q;
has been modified to reduce off-target effects; or
is a mutated hADAR2d comprising mutation T375G/S, N473D, or both, or a mutated hADAR1d comprising corresponding mutations.

9. The method of claim 1 , wherein said Cpf1 nickase protein and optionally said adenosine deaminase protein or catalytic domain thereof comprise one or more heterologous nuclear localization signal(s) (NLS(s)).

10. The method of claim 1 , wherein said method comprises, determining said target sequence of interest and selecting said adenosine deaminase protein or catalytic domain thereof which most efficiently deaminates said Adenine present in said target sequence.

11. The method of claim 1 , wherein said Cpf1 nickase protein is obtained from a Cpf1 nuclease derived from a bacterial species selected from the group consisting of Francisella tularensis, Francisella novicida, Prevotella albensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella sp., Acidaminococcus sp., Lachnospiraceae bacterium, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens and Porphyromonas macacae, Succinivibrio dextrinosolvens, Prevotella disiens, Flavobacterium branchiophilum, Helcococcus kunzii, Eubacterium sp., Microgenomates ( Roizmanbacteria ) bacterium, Flavobacterium sp., Prevotella brevis, Moraxella caprae, Bacteroidetes oral, Porphyromonas cansulci, Synergistes jonesii, Prevotella bryantii, Anaerovibrio sp., Butyrivibrio fibrisolvens, Candidatus Methanomethylophilus, Butyrivibrio sp., Oribacterium sp., Pseudobutyrivibrio ruminis and Proteocatella sphenisci.

12. The method of claim 11 , wherein said Cpf1 nickase protein
is a Francisella novicida Cpf1 nickase and recognizes a PAM sequence of TTN, wherein N is A/C/G or T, or said Cpf1 nickase protein is a Prevotella albensis Cpf1p, Lachnospiraceae bacterium Cpf1 or Acidaminococcus sp. Cpf1 nickase and recognizes a PAM sequence of TTTV, wherein V is A/C or G; or
has been modified and recognizes an altered PAM sequence.

13. The method of claim 1 , wherein said target locus of interest is within a cell, within an animal, within a plant, or comprised in a DNA molecule in vitro.

14. The method of claim 13 , wherein said cell is a eukaryotic cell, a non-human animal cell, a human cell, or a plant cell.

15. The method of claim 1 , wherein said components (a), (b) and (c) are delivered to said cell as a ribonucleoprotein complex, or as one or more polynucleotide molecules.

16. The method of claim 15 , wherein said one or more polynucleotide molecules comprise one or more mRNA molecules encoding components (a) and/or (c), or are comprised within one or more vectors.

17. The method of claim 16 , wherein said one or more polynucleotide molecules comprise one or more regulatory elements operably configured to express said Cpf1 nickase protein, said guide molecule, and said adenosine deaminase protein or catalytic domain thereof, optionally wherein said one or more regulatory elements comprise inducible promoters.

18. The method of claim 15 , wherein said one or more polynucleotide molecules or said ribonucleoprotein complex are delivered via particles, vesicles, or one or more viral vectors.

19. The method of claim 18 , wherein said particles comprise a lipid, a sugar, a metal a protein, or lipid nanoparticles.

20. The method of claim 18 , wherein said vesicles comprise exosomes or liposomes.

21. The method of claim 18 , wherein said one or more viral vectors comprise one or more of adenovirus, one or more lentivirus or one or more adeno-associated virus.

22. An engineered, non-naturally occurring system suitable for modifying an Adenine in a target locus of interest, comprising
a) a guide molecule which comprises a guide sequence linked to a direct repeat sequence, or a nucleotide sequence encoding said guide molecule;
b) a Cpf1 nickase protein, or a nucleotide sequence encoding said Cpf1 nickase protein; and
c) an adenosine deaminase protein or catalytic domain thereof, or a nucleotide sequence encoding said adenosine deaminase protein or catalytic domain thereof;
wherein said adenosine deaminase protein or catalytic domain thereof is covalently or non-covalently linked to said Cpf1 nickase protein or said guide molecule or is adapted to link thereto after delivery;
wherein said guide sequence is capable of hybridizing with a target sequence comprising an Adenine on a first DNA

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College

Inventors

• Zhang Feng
• Cox David Benjamin Turitz
• Gootenberg Jonathan
• Abudayyeh Omar O
• Zetsche Bernd
• Strecker Jonathan

Classifications

• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title

• A61K48/00

  Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy · CPC title

• C12N9/22

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

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title