Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
Department of Bioinformatics, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
BMC Genomics. 2022 Feb 15;23(1):135. doi: 10.1186/s12864-022-08359-1.
Single-cell CRISPR screens are powerful tools to understand genome function by linking genetic perturbations to transcriptome-wide phenotypes. However, since few cells can be affordably sequenced in these screens, biased sampling of cells could affect data interpretation. One potential source of biased sampling is clonal cell expansion.
Here, we identify clonal cells in single cell screens using multiplexed sgRNAs as barcodes. We find that the cells in each clone share transcriptional similarities and bear segmental copy number changes. These analyses suggest that clones are genetically distinct. Finally, we show that the transcriptional similarities of clonally expanded cells contribute to false positives in single-cell CRISPR screens.
Experimental conditions that reduce clonal expansion or computational filtering of clonal cells will improve the reliability of single-cell CRISPR screens.
单细胞 CRISPR 筛选是通过将遗传扰动与全转录组表型联系起来，从而了解基因组功能的强大工具。然而，由于在这些筛选中只能经济地测序少数细胞，因此细胞的偏倚采样可能会影响数据解释。偏倚采样的一个潜在来源是克隆细胞的扩增。
在这里，我们使用多重 sgRNA 作为条形码来鉴定单细胞筛选中的克隆细胞。我们发现每个克隆中的细胞具有转录相似性，并具有片段拷贝数变化。这些分析表明克隆是遗传上不同的。最后，我们表明克隆扩增细胞的转录相似性导致单细胞 CRISPR 筛选中的假阳性。
减少克隆扩增的实验条件或对克隆细胞进行计算过滤将提高单细胞 CRISPR 筛选的可靠性。
