Pooled CRISPRi screen with scRNA-seq to knockdown 7 transcription factors in k562 cells

To systematically investigate the gene regulatory networks in K562 cells, we performed CROP-seq—a pooled CRISPRi screen combined with single-cell RNA-seq—targeting seven transcription factors. To enhance gRNA recovery, we incorporated the Single Cell 5' CRISPR Screening Assay, which enables direct capture of gRNA sequences on the beads and generates a gRNA count matrix. Overall design: After designing and ordering 26 gRNAs (three targeting each gene plus five non-targeting controls) in a pooled format, we generated the gRNA library following the CROP-seq protocol. Next, we performed next-generation sequencing (NGS) to assess the quality of both the plasmid and lentiviral gRNA libraries. Upon confirming that both libraries passed quality control, we separately transduced wild-type K562 cells with dCas9 and the gRNA lentiviral library. Following FACS sorting and subsequent culturing, the cells were processed for single-cell RNA-seq.