Using the Microsensor system for enrichment of DROSHA-KO cells in CRISPR-Cas9 experiments. (A,C) FACS results of DROSHA-KO experiments in HEK293T cells, using pairs of sgRNA, sgDE3 and sgDE6 in A, and sgDE2 and sgDE7 in C. FACS analysis was performed. It displayed ZsGreen levels on the y-axis and mCherry levels on the x-axis. Cells transfected with Microsensor-minus and control sgRNA served as controls to outline the fluorescence profile potentially indicative of successful DROSHA-KO in cells transfected with Microsensor-plus and DROSHA-KOsgRNAs. (B,D) PCR analysis of genomic DNA from DROSHA-KO experiments. The PCR targeted genomic DNA from cells involved in the DROSHA-KO experiments (A,C). For the sgDE3 and sgDE6 pairs, the expected PCR product size for wild-type cells is 616 bp, while for DROSHA-KO cells, it is ∼458 bp. For sgDE2 and sgDE7 pairs, the wild-type size is 269 bp, while the KO size is ∼189 bp. (E,G) Single-cell PCR analysis of genomic DNA from DROSHA-KO experiments. This analysis involves genomic DNA extracted from individual cells. PCR amplification of this DNA showed a wild-type ladder with DNA fragments of 616 bp. (F,H) Pie chart of edited cells. These pie charts illustrate the proportion of cells containing the KO-band, as identified from the PCR experiments described in E and G. (I,J) Sequencing analysis of DROSHA-KO cells. DNA regions containing mutations from DROSHA-KO cells (clones 13A6 in I and 35-1H in J) were amplified via PCR. The amplified DNA was either sequenced directly by Sanger sequencing (in I) or cloned into a TOPO-TA plasmid for further Sanger sequencing (in J). Each line represents one sequencing result from each TOPO-TA plasmid clone. Deletion regions are marked by dashed lines. (K,L) Western blot analysis performed on DROSHA-KO cells (13A6 in K and 35-1H in L) and wild-type cells, this analysis aimed to detect differences in protein expression. (M,N) Quantification of miRNA expression. The expression levels of miR-16-5p and miR-30a-5p were quantified using qPCR in both wild-type and DROSHA-KO cells (13A6 in M and 35-1H in N). Results were normalized to the expression level of U6 snRNA.
