Compositions of and methods for in vitro viral genome engineering
Inventors
Cady, Kyle C. • Barbu, E. Magda • DiPetrillo, Christen G.
Assignees
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Publication Number
US-10837004-B2
Publication Date
2020-11-17
Expiration 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.
Core Innovation
The invention relates to in vitro nucleic-acid engineering in which a region of an isolated nucleic acid or first viral genome is digested using an endonuclease or RNA-guided nuclease, and at least one fragment of the digested nucleic acid is assembled with repair nucleic acid to generate an engineered viral genome or recombinant nucleic acid sequence. The second viral genome comprises at least one modification compared to the first viral genome, and the resulting nucleic acid is introduced into a host cell capable of producing viral particles to produce engineered recombinant phage.
The document describes RNA-guided nuclease implementations including Type II Cas9 and Cas9, with guiding RNA configurations such as a chimeric gRNA and crRNA plus a tracrRNA. It also discloses fully in vitro viral genome or nucleic acid engineering using RNA-guided Cas9 digestion followed by in vitro assembly to form a recombinant viral genome.
For assembling, the document provides an in vitro single-vessel assembly concept in which insertion of a DNA fragment into a digested nucleic acid is performed using a component mixture that includes an isolated 5′ to 3′ exonuclease lacking 3′ exonuclease activity, an isolated non-strand-displacing DNA polymerase with 3′ exonuclease activity, an isolated ligase, and a dNTP mixture under conditions effective for insertion. The invention further expands to generating recombinant phage that express two or more payloads selected from DNase, exopolysaccharide (EPS) depolymerase, and surfactant phenol soluble modulin (PSM).
Claims Coverage
The provided independent claims are clm-00001 and clm-00018. Together they cover two inventive features: generating an engineered viral genome from a first viral genome via endonuclease digestion and repair-nucleic-acid assembly for producing recombinant phage expressing multiple payloads, and performing a single-vessel in vitro assembly in which an RNA-guided nuclease-digested nucleic acid receives a DNA fragment using a defined exonuclease/polymerase/ligase/dNTP component mixture.
Recombinant phage genome generation by in vitro endonuclease digestion and fragment assembly into an engineered viral genome
Providing a first viral genome from a first phage; in vitro digesting a region of the first viral genome using an endonuclease; and assembling at least one fragment of the digested 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, where introduction into a host cell produces viral particles with two or more payloads selected from DNase, EPS depolymerase, and surfactant phenol soluble modulin (PSM); and introducing the second viral genome into a host cell capable of producing viral particles to produce the engineered recombinant phage.
Single-vessel in vitro recombinant nucleic acid assembly after RNA-guided nuclease digestion
Providing a nucleic acid; in vitro digesting a region of the nucleic acid using an RNA-guided nuclease; and assembling a recombinant nucleic acid by insertion of a DNA fragment into the digested nucleic acid, where assembling is performed in vitro in a single vessel with a mixture comprising an isolated 5′ to 3′ exonuclease lacking 3′ exonuclease activity, an isolated non-strand-displacing DNA polymerase with 3′ exonuclease activity, an isolated ligase, and a mixture of dNTPs under conditions effective for insertion to form the recombinant nucleic acid sequence.
The claim set is grounded in two core inventive activities: generating an engineered viral genome from a first viral genome via endonuclease digestion and repair-nucleic-acid fragment assembly for producing recombinant phage expressing multiple payload classes, and performing a single-vessel in vitro assembly in which an RNA-guided nuclease-digested nucleic acid receives a DNA fragment using a defined exonuclease/polymerase/ligase/dNTP component mixture.
Stated Advantages
Engineered recombinant phage produces viral particles with two or more payloads selected from DNase, EPS depolymerase, and PSM.
Engineered viral genomes comprise at least one modification compared to the first viral genome for producing engineered recombinant phage.
Recombinant nucleic acid assembly is performed in vitro in a single vessel to form a recombinant nucleic acid sequence.
Precision and speed.
Applicability to toxic and non-genetically tractable viruses.
Avoidance of PCR-based error-prone correction for large plasmids.
Retention of native genome modifications.
Documented Applications
Engineering bacteriophage (e.g., LUZ19 and derivatives) for proof-of-concept gene replacement and engineered phage expressing multiple payloads.
Host-range expansion and genus-level host-range “collapsing” via iterative point-mutation engineering of bacteriophage variants.
Engineered phage modifications for improved replication and early biofilm disruption via gp34 L55Δ.
Iterative multi-property engineering combining host-range and biofilm-disrupting payloads, including EPS depolymerases and PSM/PSM-family proteins.
Engineered phage payloads for antimicrobial payloads to prevent phage-resistance and sensitize to antibiotics, including lysins and expression of a species-specific antimicrobial protein payload (PyoS5).
Engineered bacteriophages to improve multiple viral properties, including host range, lytic cycle and burst size, immune-related properties, biofilm dispersion, antibiotic sensitization, and resistance prevention.
Producing engineered recombinant phage particles that express two or more payloads selected from DNase, EPS depolymerase, and one or more PSMs.
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