Compositions of and methods for in vitro viral genome engineering

Patent metadata

Field	Value
Publication number	US-2016186147-A1
Application number	US-201514970458-A
Country	US
Kind code	A1
Filing date	Dec 15, 2015
Priority date	Dec 16, 2014
Publication date	Jun 30, 2016
Grant date	—

Abstract

The present disclosure relates to a method of in vitro engineering of nucleic acids. This disclosure further relates to in vitro engineering of viral genomes and to the improvement of viral properties by in vitro genomic engineering of viral genomes. Specifically, the disclosure relates to in vitro viral genomic digestion using RNA-guided Cas9, the assembly of a recombinant genome by the insertion of a DNA or RNA fragment into the digested viral genome and transformation of a host cell with the recombinant genome. This method also related to in vitro engineering for error correction of nucleic acids.

First claim

1 . An engineered virus comprising an engineered viral nucleic acid capable, upon introduction into a host cell, of producing non-naturally occurring viral particles with two or more improved viral properties compared to the viral particles produced by introduction of the non-engineered viral nucleic acid into a host cell.

2 . The engineered virus of claim 1 , wherein the produced viral particles have at least three improved viral properties.

3 . The engineered virus of claim 1 , wherein each improved viral property is selected from the group consisting of host range, viral lytic cycle, adsorption, attachment, injection, replication and assembly, lysis, burst size, immune evasion, immune stimulation, immune deactivation, biofilm dispersion, bacterial phage resistance, bacterial antibiotic sensitization, modulation of virulence factors, and targeted host genome digestion or editing.

4 . The engineered virus of claim 1 , wherein the engineered viral nucleic acid is an engineered viral genome.

5 . The engineered virus of claim 4 , wherein the engineered viral genome is an engineered bacteriophage genome.

6 . The engineered virus of claim 5 , wherein at least one of the improved properties is host range.

7 . The engineered virus of claim 1 , wherein each improved viral property is the result of at least one modification in the engineered viral nucleic acid.

8 . The engineered virus of claim 7 , wherein at least one improved viral property is the result of at least two modifications in the engineered viral nucleic acid.

9 . The engineered virus of claim 7 , wherein the at least one modification in the engineered viral nucleic acid are the result of a single engineering step.

10 . The engineered virus of claim 7 , wherein the at least one modification in the engineered viral nucleic acid are the result of iterative engineering steps.

11 . The engineered virus of claim 7 , wherein at least one of the modifications is within a nucleic acid sequence encoding an amino acid sequence having at least 85% identity to SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:5, SEQ ID NO:48, or SEQ ID NO:49.

12 . The engineered virus of claim 4 , wherein the engineered viral genome comprises all or a portion of a viral genome having at least 95% identity to the LUZ19 genome.

13 - 24 . (canceled)

25 . A method for generating an engineered virus of interest having two or more desired viral properties comprising:
(a) providing a first viral genome; and (b) generating an engineered viral genome by combining at least one fragment of the first viral genome with at least one repair nucleic acid molecule to generate a second viral genome comprising at least one modification compared to the first viral genome; wherein, the second viral genome, upon being introduced into a host cell, is capable of producing viral particles with two or more improved properties.

26 . The method of claim 25 , further comprising:
(c) repeating steps (a)-(b) in one or more iterations.

27 . The method of claim 25 , wherein each improved viral property is selected from the group consisting of host range, viral lytic cycle, adsorption, attachment, injection, replication and assembly, lysis, burst size, immune evasion, immune stimulation, immune deactivation, biofilm dispersion, bacterial phage resistance, bacterial antibiotic sensitization, modulation of virulence factors, and targeted host genome digestion or editing.

28 . The method of claim 25 , wherein generating an engineered viral genome in step (b) comprises:
(1) in vitro digestion of a region of the first viral genome using an endonuclease; and (2) assembling at least one fragment of the digested first viral genome with at least one repair nucleic acid molecule.

29 . The method of claim 28 , wherein the first viral genome is isolated from viral particles.

30 . The method of claim 28 , wherein the first viral genome or the at least one repair nucleic acid molecule is synthesized de novo.

31 . The method of claim 30 , wherein de novo synthesis comprises combining chemically synthesized nucleic acid molecules, PCR-amplified nucleic acid sequences, digested fragments of isolated nucleic acid molecules, or any combination thereof.

32 . The method of claim 30 , wherein the first viral genome or the at least one repair nucleic acid molecule is amplified prior to in vitro digestion.

33 . The method of claim 26 , wherein the first viral genome is at least 3 kb, at least 10 kb, at least 18 kb, at least 25 kb, or at least 30 kb.

34 . The method of claim 28 , wherein the assembly is performed in vitro or in vivo.

35 . The method of claim 34 , wherein the assembly is performed in vitro with a mixture comprising:
(a) an isolated 5′ to 3′ exonuclease that lacks 3′ exonuclease activity; (b) an isolated non-strand-displacing DNA polymerase with 3′ exonuclease activity, or a mixture of said DNA polymerase with a second DNA polymerase that lacks 3′ exonuclease activity; (c) an isolated ligase; and (d) a mixture of dNTPs, under conditions that are effective for insertion of the fragment into the digested viral nucleic acid to form a recombinant nucleic acid comprising the engineered viral genome.

36 . The method of claim 28 , wherein the endonuclease is an RNA-guided nuclease.

37 . The method of claim 36 , further comprising at least one guiding RNA.

38 . The method of claim 37 , wherein the RNA-guided nuclease is Cas9 or a Cas9 derived enzyme, and wherein the at least one guiding RNA comprises 1) a chimeric gRNA or 2) a crRNA and tracrRNA.

39 . The method of claim 28 , wherein the endonuclease is heat inactivated or removed prior to assembly.

40 . The method of claim 28 , wherein the in vitro digestion further comprises spermidine.

41 . The method of claim 28 , further comprising transforming the engineered viral genome into a host cell.

42 . The method of claim 28 , further comprising using an in vitro packaging kit for packaging of the engineered viral genome into viral particles.

43 . An engineered virus generated by any of the methods in claims 26 - 46 .

44 . The engineered viruses of claim 47 , wherein the engineered virus is the engineered virus of any of claims 1 - 12 and 25 .

45 . A kit for engineering viral nucleic acid molecules comprising:
(a) purified recombinant RNA-guided nuclease; (b) an isolated 5′ to 3′ exonuclease that lacks 3′ exonuclease activity; (c) an isolated non-strand-displacing DNA polymerase with 3′ exonuclease activity, or a mixture of said DNA polymerase with a second DNA polymerase that lacks 3′ exonuclease activity; and (d) an isolated ligase.

46 . The kit of claim 45 , further comprising one or more of:
(1) a crowding agent; (2) a mixture of dNTPs; and (3) a suitable buffer.

47 . The kit of claim 45 , further comprising custom-designed guide RNAs.

48 . The kit of claim 45 , further comprising custom-designed synthesized nucleic acid molecules to serve as the inserted DNA fragment in an assembly reaction.

49 . The kit of claim 45 , further comprising competent host cells for transformation.

50 . The kit of claim 45 , further comprisin

Assignees

• Synthetic Genomics Inc

Inventors

• Cady Kyle C
• Barbu E Magda
• Dipetrillo Christen G

Classifications

• C12N2795/14121

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C12N2795/10321

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C12N2795/10222

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• C12N7/00 Primary

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N2795/14122

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title