sgrna targeting exon 5  (Addgene inc)

Journal: Human genetics

Article Title: oFlowSeq: a quantitative approach to identify protein coding mutations affecting cell type enrichment using mosaic CRISPR-Cas9 edited cerebral organoids

doi: 10.1007/s00439-023-02534-4

Figure Lengend Snippet: Hypothetical schematic of the oFlowSeq method to identify cell types affected by KCTD13 mutations in CRISPR-edited cerebral organoids. A Schematic of oFlowSeq method using CRISPR-Cas9 editing of the KCTD13 gene in iPSCs to differentiate mosaic cerebral organoids. These mosaic organoids, which comprised of different cell types with different KCTD13 genotypes, were dissociated into single cells for FACS into seven sorted pools of cells. B Hypothetical schematic of cell counts for a deleterious mutation in KCTD13 that enriches for Nestin+ cells while depleting TRA-1–60+ cells, with an OR > 1 representing an enrichment; OR < 1 representing a depletion; and OR = 1 representing no changes in cell proportions. C Hypothetical schematic of cell counts for a deleterious mutation in KCTD13 that enriches for Nestin+ cells while depleting NeuN+ cells

Article Snippet: We used an sgRNA targeting the second exon of KCTD13 (chr16:29923312–29923331; sequence 5′-UGAG GAUUGU AC CAA AGU GA-3′) and Cas9 2NLS nuclease from Synthego, and incubated 3 μl of 100 μM sgRNA with 2 μl of 20 μM Cas9 nuclease at room temperature for 10 min prior to nucleofection.

Techniques: CRISPR, Mutagenesis

Journal: Human genetics

Article Title: oFlowSeq: a quantitative approach to identify protein coding mutations affecting cell type enrichment using mosaic CRISPR-Cas9 edited cerebral organoids

doi: 10.1007/s00439-023-02534-4

Figure Lengend Snippet: Odds ratios to correlate mutations with cell type proportions. A Hypothetical expected distribution of ORs for benign mutations that do not affect cell type proportions (in blue). B Hypothetical expected distribution of ORs for benign mutations that do not affect cell type proportions (in blue), and deleterious mutations that increase cell type proportions (in pink). C Observed density distributions of short mutations (red), long mutations (blue), and all mutations (black) for NeuN, Nestin, and TRA-1–60 positively stained cells. D Observed heatmap representation of the scaled odds ratios for each of the 19 synthetic mutations in KCTD13 for TRA-1–60, Nestin, and NeuN positively stained cells

Article Snippet: We used an sgRNA targeting the second exon of KCTD13 (chr16:29923312–29923331; sequence 5′-UGAG GAUUGU AC CAA AGU GA-3′) and Cas9 2NLS nuclease from Synthego, and incubated 3 μl of 100 μM sgRNA with 2 μl of 20 μM Cas9 nuclease at room temperature for 10 min prior to nucleofection.

Techniques: Staining