Methods and compositions for generating a deletion library and for identifying a defective interfering particle (dip)

Patent metadata

Field	Value
Publication number	US-2024035021-A1
Application number	US-202318171578-A
Country	US
Kind code	A1
Filing date	Feb 20, 2023
Priority date	Dec 14, 2016
Publication date	Feb 1, 2024
Grant date	—

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 ae human immunodeficiency virus (HIV) deletion mutants, e.g., interfering, conditionally replicating, HIV deletion mutants, and related constructs.

First claim

1 . A method of generating a deletion library, comprising:
(a) 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; (b) contacting the population of transposon-inserted circular target DNAs with the sequence specific DNA endonuclease to generate a population of cleaved linear target DNAs; (c) contacting the population of cleaved linear target DNAs with one or more exonucleases to generate a population of deletion DNAs; and (d) circularizing the deletion DNAs to generate a library of circularized deletion DNAs.

2 . The method of claim 1 , wherein the circular target DNAs are plasmids that comprise a viral genome.

3 . The method of claim 2 , wherein the method further comprises introducing members of the library of circularized deletion DNAs into mammalian cells, and assaying for viral infectivity.

4 . The method of claim 2 , wherein the method further comprises sequencing members of the library of circularized deletion DNAs to identify defective interfering particles (DIPs).

5 . The method of claim 1 , wherein the sequence specific DNA endonuclease is selected from: a meganuclease, a CRISPR/Cas endonuclease, a zinc finger nuclease, or a TALEN.

6 . The method of claim 1 , wherein the method comprises inserting a barcode sequence prior to or simultaneous with step (d).

7 . The method of claim 1 , wherein the one or more exonucleases comprises T4 DNA polymerase.

8 . The method of claim 1 , wherein the one or more exonucleases comprises a 3′ to 5′ exonuclease and a 5′ to 3′ exonuclease.

9 . The method of claim 1 , wherein the one or more exonucleases comprises RecJ.

10 . The method of claim 1 , wherein the step of contacting the population of cleaved linear target DNAs with one or more exonucleases is performed in the presence of a single strand binding protein (SSB).

11 . The method of claim 1 , wherein the transposon cassette comprises a first recognition sequence positioned at or near one end of the transposon cassette and a second recognition sequence positioned at or near the other end of the transposon cassette.

12 . The method of claim 1 , further comprising, prior to step (a), circularizing a population of linear DNA molecules to generate said population of circular target DNAs.

13 . The method of claim 12 , wherein the population of linear DNA molecules comprises one or more PCR products, one or more linear viral genomes, and/or one or more restriction digest products.

14 . The method of claim 1 , further comprising introducing members of the library of circularized deletion DNAs into mammalian cells.

15 . The method of claim 1 , further comprising generating from the library of circularized deletion DNAs, at least one of: linear double stranded DNA (dsDNA) products, linear single stranded DNA (ssDNA) products, linear single stranded RNA (ssRNA) products, and linear double stranded RNA (dsRNA) products.

16 . The method of claim 15 , further comprising introducing said linear dsDNA products, linear ssDNA products, linear ssRNA products, and/or linear dsRNA products into mammalian cells.

17 . The method of claim 12 , wherein the inserting of step (a) comprises inserting a transposon cassette into the population of circular target viral DNAs, wherein the transposon cassette comprises the target sequence for the sequence specific DNA endonuclease, and wherein said generated population of sequence-inserted viral DNAs is a population of transposon-inserted viral DNAs.

Assignees

• The J David Gladstone Inst
• Univ California

Inventors

• Weinberger Leor S
• Notton Timothy J

Classifications

• C12N15/1082 Primary

  Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title