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Lentiviral co-packaging mitigates the effects of intermolecular recombination and multiple integrations in pooled genetic screens

David Feldman, Avtar Singh, Anthony J. Garrity, Paul C. Blainey
doi: https://doi.org/10.1101/262121
David Feldman
1Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA
2The Broad Institute of MIT and Harvard, Cambridge, MA, USA
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Avtar Singh
2The Broad Institute of MIT and Harvard, Cambridge, MA, USA
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Anthony J. Garrity
2The Broad Institute of MIT and Harvard, Cambridge, MA, USA
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Paul C. Blainey
2The Broad Institute of MIT and Harvard, Cambridge, MA, USA
3Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
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  • For correspondence: pblainey@broadinstitute.org
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Abstract

Lentiviral vectors are widely used for functional genomic screens, enabling efficient and stable transduction of target cells with libraries of genetic elements. Unfortunately, designs that rely on integrating multiple variable sequences, such as combinatorial perturbations or perturbations linked to barcodes, may be compromised by unintended consequences of lentiviral packaging. Intermolecular recombination between library elements and integration of multiple perturbations (even at limiting virus dilution) can negatively impact the sensitivity of pooled screens. Here, we describe a simple approach to prevent recombination between lentiviral vectors containing multiple linked variable elements, such as the recently reported CRISP-seq, Perturb-seq, and Mosaic-seq designs. We show that modifying the packaging protocol by diluting the perturbation library with a carrier plasmid increases the fraction of correct, single integrations from <60% to >90%, at the cost of reducing titer by 100-fold.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted February 08, 2018.
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Lentiviral co-packaging mitigates the effects of intermolecular recombination and multiple integrations in pooled genetic screens
David Feldman, Avtar Singh, Anthony J. Garrity, Paul C. Blainey
bioRxiv 262121; doi: https://doi.org/10.1101/262121
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Lentiviral co-packaging mitigates the effects of intermolecular recombination and multiple integrations in pooled genetic screens
David Feldman, Avtar Singh, Anthony J. Garrity, Paul C. Blainey
bioRxiv 262121; doi: https://doi.org/10.1101/262121

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