![]() Examples of clonal (left) and non-clonal (right) cells. ![]() Overview of single-cell CRISPR screen performed in this study (~ 20 k sgRNAs, ~ 50 k cells sequenced, ~ 30 sgRNAs per cell). Multiplexed sgRNAs serve as a clonal barcode. In sum, this approach can be used to improve the quality and interpretation of single-cell CRISPR screens, and it can allow clonal lineage information to be derived from these data. Finally, we identify segmental copy number changes by comparing the transcriptomes of clonal cells, and these data suggest that clones are also genetically distinct. ![]() We identified distinct populations of clonal cells, and we show that removal of clonal cells significantly reduces false discovery. To test this approach, we performed a single-cell screen in breast cancer cells. We developed a computational strategy to identify clones based on multiplexed sgRNA barcodes in sequenced cells. We reason that the combination of sgRNAs in a cell can serve as a barcode to track clonal cells. Single-cell CRISPR screens have introduced multiple sgRNAs per cell to increase throughput. Quantification of clonal cells is necessary to assess the clonality in single cell screens. However, in single-cell CRISPR screens, a relatively smaller number of cells can be affordably sequenced, which increases the risk of clonal expansion. Traditional bulk CRISPR screens analyze millions of cells, which limits clonal expansion artifacts. Excessive clonal cell expansion could potentially bias cell-based screening by oversampling highly proliferative clones, thereby increasing false signals or decreasing true signals. Cancer cells are heterogeneous, and distinct clones have genetic features that facilitate proliferation. One such parameter is clonal cell expansion. However, standard procedures for single-cell CRISPR screens have not been systematically evaluated, and how certain experimental parameters affect data interpretation remain understudied. Recently, single-cell CRISPR screens have been developed to link genetic perturbations with high content transcriptome-wide phenotypes. Traditional CRISPR screens rely on a phenotypic selection step such as proliferation. CRISPR screens are powerful genetic tools to study the function of genes and regulatory elements genome-wide.
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