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

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.

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.