GiRAFR improves gRNA detection and annotation in single cell CRISPR screens

Novel single cell RNA-seq analysis combined with CRISPR screens enables the high- throughput characterization of transcriptional changes caused by genetic perturbations. Dedicated software to annotate CRISPR guide RNA (gRNA) libraries and associate them with single cell transcriptomes are however lacking. Here, we generated a CRISPR droplet sequencing dataset. We demonstrate that the current default tool fails to detect mutant gRNAs. We therefore developed GiRAFR, a pysam-based software tool to characterize intact and mutant gRNAs. We show that mutant gRNAs are dysfunctional, and failure to detect and annotate them leads to an inflated estimate of the number of untransformed cells as well as an underestimated multiplet frequency. These findings are mirrored in publicly available datasets, where we find that up to 34 % of cells are transduced with a mutant gRNA. Applying GiRAFR hence stands to improve the annotation and quality of single cell CRISPR screens. Overall design: We transformed A549 cells expressing a tamoxifen-inducible Cas9 with a lentiviral pool for expression of 120 gRNAs at low multiplicity of infection. After stringent selection using puromycin and 2 days of Cas9 induction, cells were allowed to grow for another 5 days.

新型单细胞RNA测序（single cell RNA-seq）分析结合CRISPR筛选技术，可实现遗传扰动引发的转录组变化的高通量表征。然而，目前尚缺乏可用于注释CRISPR向导RNA（guide RNA, gRNA）文库并将其与单细胞转录组进行关联的专用软件。本研究构建了一套CRISPR液滴测序数据集，并证实当前主流的默认工具无法检测突变型gRNA。为此，我们开发了基于pysam的专用软件工具GiRAFR，用于对完整型与突变型gRNA开展表征分析。本研究证实，突变型gRNA会丧失功能；若未对其进行检测与注释，会导致未转化细胞数量的估计值偏高，同时低估多重转导细胞的频率。该现象在公开数据集同样存在：本研究发现，最高可达34%的细胞被突变型gRNA转导。因此，应用GiRAFR可有效提升单细胞CRISPR筛选的注释质量与整体数据品质。
实验整体设计：将携带120条gRNA的慢病毒文库以低感染复数转导至表达他莫昔芬诱导型Cas9的A549细胞中。经嘌呤霉素严格筛选与2天的Cas9诱导后，将细胞继续培养5天。