Fig 1.
Workflows for arrayed screening in microplate format.
(A) Reverse transfection of cells with siRNA. (B) Potential workflow for the reverse transfection of synthetic CRISPR reagents. crRNAs are first pre-spotted to plates and tracrRNA is added in serum free media. The complex is then incubated with lipid-based transfection reagents prior to the addition of cells.
Fig 2.
Cellular effects of modulating GMNN with synthetic CRISPR reagents or siRNA.
(A) Representative images illustrating alterations in nuclear size (Hoechst staining; lower panels) and GMNN protein levels by immunofluorescence (upper panels). (B) Quantitation of changes in nuclear count, nuclear area, and GMNN protein levels (n = 8 per condition; median effect and standard deviation are represented).
Fig 3.
Selection and characterization of HCT-116 Cas9 clones.
(A) Clones transfected with synthetic crRNA targeting GMNN exhibit increased nuclear area as compared to the parental HCT-116 Cas9 population. All exhibited a significant increase for crGMNN versus non-targeting control (p < 0.05) (B) Western blot indicates a greater decrease in GMNN protein for clones transfected with crGMNN versus parental HCT-116 Cas9 cells. Clones also exhibit greater levels of Cas9. (C) NGS sequencing of the GMNN target region in transfected cells (Clone-A) indicates >80% editing of GMNN DNA. (D) Effects of crRNAs targeting control genes known to affect nuclear area in HCT-116 Cas9 polyclonal and clonal populations. Bars represent the average and standard deviation of four replicates. The dashed line indicates five standard deviations above non-targeting control. Data for additional genes can be found in S2 Fig.
Fig 4.
Various relationships among crRNA and siRNA screen data.
(A) Biological replicates of a synthetic CRISPR screening experiment. Each dot indicates the value derived from a single crRNA assayed in separate experiments. (B) Increased nuclear area exhibits a strong negative correlation with nuclear count. Points correspond to values from individual wells. (C-D). Comparison of the effect of different guides (C) or siRNA (D) targeting the same gene on measured nuclear area. “Sets” are the average activity of randomly selected pairs of guides or siRNAs targeting the same gene. There are two pairs per gene given that each gene has 4 corresponding crRNA or siRNA reagents. The correlation between any randomly selected single crRNA targeting the same gene was also greater than that for siRNA (r = 0.45 versus r = 0.18; S4 Fig).
Fig 5.
Evaluation of screen results from synthetic CRISPR and siRNA screens of ubiquitin system genes.
(A) RSA reveals many more significant hits (FDR < 0.05) for CRISPR relative to siRNA screening. (B) Genes identified through CRISPR screening with a RSA corrected p-values < 0.001 and corresponding values from siRNA screening. “OTP siRNA” refers to results from screening of the Dharmacon On-Target Plus siRNA library. “SilSelect siRNA” refers to results from screening the Ambion Silencer Select siRNA library. “All siRNA” refers to results obtained after combing data from both siRNA screens. *Refers to crRNA screen hits that appear to be supported by siRNA data. (C) Genes with RSA corrected p-value < 0.001 in the CRISPR screen are associated with progression through the cell cycle.