Co-packaging to mitigate intermolecular recombination

A method of preparing one or more packaging cells capable of producing a viral expression library the method comprising transfecting one or more packaging cells with a system comprising a pooled set of payload polynucleotides, each payload polynucleotide comprising or encoding at least a genetic perturbation in association with a barcode that identifies the genetic perturbation(s), a multiplicity of carrier polynucleotides that are heterologous to the payload polynucleotides and do not have or encode at least a genetic perturbation in association with a barcode that identifies the genetic perturbation(s), and one or more packaging polynucleotides, wherein the system comprises a 5:1 weight ratio or greater mixture of the carrier polynucleotides to the pooled set of the payload polynucleotides, wherein the packaging cells are capable of producing a viral expression library comprising viral particles, wherein each one of the pooled set of the payload polynucleotides is comprised in at least one of the viral particles of the viral expression library, wherein the packaging cells are sufficiently capable of packaging no more than one payload polynucleotide per viral particle such that target cells transduced with the viral expression library have reduced recombination activity, or template switching activity, or multiple integration activity, as compared to counterpart packaging cells transfected with a mixture comprising the payload polynucleotides in the absence of the 5:1 weight ratio or greater of the carrier polynucleotides to the pooled set of the payload polynucleotides.

A packaging cell or packaging cells for producing a viral expression library comprising viral particles, wherein one or more packaging cells comprise a system comprising a pooled set of payload polynucleotides, each payload polynucleotide comprising or encoding at least a genetic perturbation associated with a barcode that identifies the genetic perturbation(s), a multiplicity of the carrier polynucleotides that are heterologous to the payload polynucleotide and do not comprise or encode at least a genetic perturbation associated with a barcode that identifies the genetic perturbation(s), and one or more packaging polynucleotides, wherein the system comprises a 5:1 weight ratio or greater mixture of the carrier polynucleotides to the pooled set of the payload polynucleotides, wherein the packaging cells are capable of producing a viral expression library comprising viral particles, wherein each one of the pooled set of the payload polynucleotides is comprised in at least one of the viral particles of the viral expression library, and wherein the packaging cells are sufficiently capable of packaging no more than one payload polynucleotide per viral particle such that target cells transduced with the viral expression library have reduced recombination activity, or template switching activity, or multiple integration activity, as compared to counterpart packaging cells transfected with a mixture comprising the payload polynucleotides in the absence of the 5:1 weight ratio or greater of the carrier polynucleotides to the pooled set of the payload polynucleotides.

A method of reducing intermolecular recombination with a lentiviral genome plasmid of interest in a pooled library, wherein the lentiviral genome plasmid of interest encodes at least a genetic perturbation associated with a barcode that identifies the genetic perturbation(s), the method comprising transfecting one or more packaging cells with a system comprising the pooled library of lentiviral genome plasmids, a multiplicity of viral carrier plasmids, wherein the lentiviral carrier plasmids are heterologous to the lentiviral genome plasmids and do not encode at least a genetic perturbation associated with a barcode that identifies the genetic perturbation(s), and one or more lentiviral packaging plasmids, wherein the system comprises a 5:1 weight ratio or greater of the lentiviral carrier plasmids to the lentiviral genome plasmids, wherein the packaging cells are capable of producing a lentiviral expression library comprising lentiviral particles, wherein each lentiviral genome plasmid of interest is comprised in at least one of the lentiviral particles, and wherein the packaging cells are sufficiently capable of packaging no more than lentiviral genome plasmid per lentiviral particle such that target cells transduced with the lentiviral expression library have reduced recombination activity, or template switching activity, or multiple integration activity, as compared to counterpart packaging cells transfected with a mixture comprising the library of the lentiviral genome plasmids in the absence of the 5:1 weight ratio or greater of the lentiviral carrier plasmids to the lentiviral genome plasmids.