重组
质粒
转导(生物物理学)
计算生物学
生物
效价
分子间力
限制
遗传学
DNA
计算机科学
化学
病毒
基因
生物物理学
工程类
分子
机械工程
有机化学
作者
David Feldman,Avtar Singh,Anthony J. Garrity,Paul C. Blainey
摘要
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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