Mosaic-seq barcode shuffling sequencing

Simultaneously detecting CRISPR-based perturbations and induced transcriptional changes in the same cell is a powerful approach to unraveling genome function. Several lentiviral approaches have been developed, some of which rely on the detection of distally located genetic barcodes as an indirect proxy of sgRNA identity. Since barcodes are often several kilobases from their corresponding sgRNAs, viral recombination-mediated swapping of barcodes and sgRNAs is feasible. Using a self-circularization-based sgRNA-barcode library preparation protocol, we measured the barcode shuffling rate while pooling the samples at different stages of library preparation. We estimate the recombination rate to be ~50% and we trace this phenomenon to the pooled viral packaging step. Recombination is random, and decreases the signalto-noise ratio of the assay. Our results suggest that alternative approaches can increase the throughput and sensitivity of single-cell perturbation assays.在同一细胞中同时检测基于成簇规律间隔短回文重复序列 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR) 的基因扰动与诱导产生的转录组变化，是解析基因组功能的强效研究手段。已有多种慢病毒介导的研究方法被开发，其中部分方法依赖于对远端遗传条形码 (genetic barcode) 的检测，以此作为单引导RNA (single guide RNA, sgRNA) 身份的间接替代标记。由于条形码通常与其对应的sgRNA相距数千碱基对，通过病毒重组介导的条形码与sgRNA交换过程具备可行性。本研究采用基于自环化的sgRNA-条形码文库制备方案，在文库制备的不同阶段对样本进行混合的同时，测定了条形码的洗牌率。我们估算该重组率约为50%，并将该现象追溯至混合病毒包装步骤。重组过程呈随机特性，会降低该检测实验的信噪比。本研究结果表明，采用替代方案可提升单细胞扰动检测实验的通量与灵敏度。