RT Journal Article
SR Electronic
T1 Lentiviral co-packaging mitigates the effects of intermolecular recombination and multiple integrations in pooled genetic screens
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 262121
DO 10.1101/262121
A1 David Feldman
A1 Avtar Singh
A1 Anthony J. Garrity
A1 Paul C. Blainey
YR 2018
UL http://biorxiv.org/content/early/2018/02/08/262121.abstract
AB 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.