Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens

Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling.

哺乳动物细胞池化筛选（pooled screens）的诸多实现方案，均依赖于将目的元件与条形码（barcode）相连，后续通过下一代测序（next generation sequencing）对条形码进行定量分析。不过已有研究报道，在慢病毒包装（lentiviral production）过程中，这些配对元件之间会出现显著的解偶联（uncoupling）现象，且元件间的间距越大，解偶联程度愈为严重。我们详细阐明，PCR扩增（PCR amplification）是另一主要的解偶联来源，且随着DNA模板分子投入量与PCR循环数的增加，解偶联现象会愈发显著。为了在使用配对元件进行检测的体系中减轻解偶联问题，我们建议：缩短元件间的间距；在PCR过程中为质粒DNA（plasmid DNA）与基因组DNA（genomic DNA）使用低且等量的DNA模板投入量；减少PCR循环总数。此外，我们还提出了一种可用于开展组合CRISPR筛选（combinatorial CRISPR screens）的载体设计方案，该方案可通过单次短读长测序实现基于条形码的精准检测，同时将解偶联程度降至最低。