Methods and compositions for generating a deletion library and for identifying a defective interfering particle (DIP)
Inventors
Weinberger, Leor S. • Notton, Timothy J.
Assignees
J David Gladstone Institutes • University of California San Diego UCSD
Publication Number
US-11584931-B2
Publication Date
2023-02-21
Expiration Date
2037-12-14
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Abstract
Provided are methods and compositions for generating a deletion library, and methods and compositions for generating and identifying a defective interfering particle (DIP). Also provided are transposon cassettes. A subject method can include: inserting a transposon cassette comprising a target sequence for a sequence specific DNA endonuclease into a population of circular target DNAs to generate a population of transposon-inserted circular target DNAs; contacting the population of transposon-inserted circular target DNAs with the sequence specific DNA endonuclease to generate a population of cleaved linear target DNAs; contacting the population of cleaved linear target DNAs with one or more exonucleases to generate a population of deletion DNAs; and circularizing the deletion DNAs to generate a library of circularized deletion DNAs. The population of circular target DNAs can include viral genomic DNA. Also provided are human immunodeficiency virus (HIV) deletion mutants, e.g., interfering, conditionally replicating, HIV deletion mutants, and related constructs.
Core Innovation
The invention provides methods and compositions for generating a deletion library from a population of circular target DNAs, including viral genomes, and for generating and identifying defective interfering particles (DIPs). The methods include inserting a transposon cassette comprising a target sequence for a sequence specific DNA endonuclease into circular target DNAs to generate transposon-inserted DNAs, cleaving them with the endonuclease to generate cleaved linear target DNAs, digesting the ends with exonucleases to create deletions, and then circularizing the deletion DNAs to form a library of circularized deletion DNAs. The invention also includes transposon cassettes engineered to facilitate these steps and HIV deletion mutants identified with these methods.
The problem solved by the invention arises from the need for additional methods and compositions for manipulating circular DNAs, such as plasmids carrying viral genomes, to generate libraries of circular DNAs with multiple deletions at varying locations. Existing methods lack efficient, high-throughput ways to produce comprehensive deletion libraries and to identify DIPs, which are viral mutants that interfere with viral replication and may have therapeutic potential. The invention offers a molecular biology method allowing control over deletion sizes and tagging of mutants for sequencing and tracking, aiding the study of viral functional elements and the discovery of therapeutic interfering particles.
Claims Coverage
The patent includes 9 independent inventive features relating to methods of generating deletion libraries and defective interfering particles (DIPs), use of exonucleases, barcode incorporation, infection screening, and novel transposon cassettes and HIV-1 deletion mutants.
Method of generating a deletion library using a transposon cassette and exonuclease digestion
A method including (a) inserting a transposon cassette with a target sequence for a specific DNA endonuclease into circular target DNAs to create transposon-inserted DNAs; (b) contacting them with the DNA endonuclease to produce cleaved linear DNAs; (c) applying exonucleases to generate deletion DNA products; and (d) circularizing these to generate a deletion library.
Method of generating and identifying defective interfering particles (DIPs)
A method comprising (a) inserting a target sequence for a DNA endonuclease into circular viral DNAs comprising viral genomes; (b) cleaving them to produce linear viral DNAs; (c) digesting with exonuclease to obtain deletion DNAs; (d) circularizing deletion DNAs to form a library; and (e) sequencing library members to identify DIPs.
Insertion of barcode sequences for mutant tracking
Inserting a barcode sequence prior to or simultaneous with circularization of deletion DNAs, enabling tracking and identification of individual deletion mutants in a library.
Use of one or more exonucleases including T4 DNA polymerase and RecJ with SSB for deletion generation
Use of exonucleases with 3′ to 5′ and 5′ to 3′ activities, specifically including T4 DNA polymerase and RecJ, optionally in the presence of single strand binding (SSB) protein, to generate controlled deletions after cleavage.
Infection and assay methods with multiplicity of infection (MOI) variation
Methods including introducing members of deletion DNA libraries into mammalian cells and assaying for viral infectivity, with high MOI (≥2) or low MOI (<1) infection schemes, culturing with replication inhibitors, addition of naive cells, and harvesting virus to identify and characterize functional DIPs.
Generation and use of transposon cassettes with flanking meganuclease recognition sequences
Transposon cassettes comprising transposase-compatible inverted terminal repeats (ITRs) flanking sequences with two recognition sites for one or two meganucleases, and including selectable marker genes such as antibiotic resistance genes, suitable for insertion into target DNAs to facilitate library generation.
Circularization of deletion DNAs from various linear DNA starting materials
Methods including, prior to insertion of target sequences, circularizing a population of linear DNA molecules such as PCR products, linear viral genomes, or restriction digest products to create the circular target DNAs used for deletion library generation.
Generating linear DNA, RNA products from deletion libraries and introducing them into cells
Generating at least one of linear double-stranded DNA, single-stranded DNA, single-stranded RNA, or double-stranded RNA products from circularized deletion DNAs and introducing these into mammalian cells for further assays.
Specific HIV-1 deletion mutants retaining all cis-acting elements and including deletions in gag and/or pol genes
HIV-1 deletion mutant constructs comprising four defined cis-acting elements (CAE1-CAE4) and deletions in gag and/or pol genes, optionally with accessory gene deletions (vif, vpr, tat, rev, vpu) and nef deletion, useful as defective interfering particles.
The independent claims collectively cover methods of generating deletion libraries via transposon insertion and exonuclease digestion, generating and identifying DIPs, insertion of barcodes for tracking, methods for assaying viral infectivity at various MOIs, use of transposon cassettes with meganuclease sites, production of linear nucleic acid products from deletion libraries, and specific HIV-1 deletion mutants preserving essential cis-elements while deleting certain genes.
Stated Advantages
The method allows control over deletion size and can tag each library member with unique barcodes, facilitating high-throughput sequencing and tracking.
The methods enable comprehensive generation of random deletions in circular DNA, including viral genomes, in a high-throughput manner.
The approach facilitates identification of cis- and trans-acting viral elements and enables discovery of defective interfering particles that may be used therapeutically.
The use of transposon cassettes and sequence specific DNA endonucleases provides a modular, customizable approach applicable to different DNA targets.
The screening methods (high and low MOI infection) allow selection for DIPs that are either efficiently mobilized by wildtype virus or non-cytopathic and transmittable.
Documented Applications
Generation of deletion libraries of circular DNAs, including viral genomes, for studying functional elements.
Identification and characterization of defective interfering particles (DIPs) from viral deletion libraries.
Screening for interfering HIV-1 deletion mutants as potential therapeutic interfering particles (TIPs).
Mapping cis- and trans-acting elements of viral genomes by assessing deletion tolerance and infectivity.
Constructing HIV-1 deletion mutants with defined deletions to study replication competence, interference with wildtype virus, and mobilization.
High multiplicity of infection (MOI) passaging of viral pools to enrich for transmissible interfering viral mutants.
Low MOI screening combined with viral replication inhibitors to select for non-cytopathic, mobilizable deletion mutants.
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