Perturb-seq is a platform for single-cell gene expression profiling of pooled CRISPR screens. Like many functional genomics platforms, Perturb-seq relies on lentiviral transduction to introduce perturbation libraries to cells. On this platform, these are barcoded sgRNA libraries. A critical consideration for performing Perturb-seq experiments is uncoupling of barcodes from linked sgRNA expression cassettes, which can occur during lentiviral transduction of co-packaged libraries due to reverse transcriptase-mediated template switching. This problem is common to lentiviral libraries designed with linked variable regions. Here, we demonstrate that recombination between Perturb-seq vectors scrambles linked variable regions separated by 2 kb. This predicts information loss in Perturb-seq screens performed with co-packaged libraries. We also demonstrate ways to address this problem and discuss best practices for single-cell screens with transcriptional readouts.
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