Pooled Screens Identify GPR108 and TM9SF2 as Host Cell Factors Critical for AAV Transduction

Figure 1. Summary of AAV CRISPR Screen Design and Optimization (A) Overview of CRISPR library generation, target cell transduction, and sgRNA enrichment. (B) Experimental design and sorting schemes for AAV2-EGFP transduction of CRISPR libraries in U-2 OS Cas9 cells. (C) Comparison of the gene-aggregated log2 ratios of sgRNA abundance in the high and low EGFP populations from the 1,200 MOI transduction (x axis) and the positive and negative populations from the 60 MOI transduction (y axis). The color of each dot is related to its expression in U-2 OS cells and the size proportional to the FDR in the comparison of the mock transduction to the plasmid library. The lines indicate the mean (solid) and three standard deviations (dotted) of all genes in each screen.

Figure 2. Two Genome-wide CRISPR Screens Identify Genes Important for AAV Transduction (A) Comparison of the gene-aggregated log2 ratios of sgRNA abundance for the high and low EGFP populations from a 1,200 MOI transduction of U-2 OS Cas9 cells harboring an 80K CRISPR library (x axis) or a 155K CRISPR library (y axis). The color of each dot is related to its expression in U-2 OS cells and the size proportional to the FDR in the comparison of the mock transduction to the plasmid library. The lines indicate the mean (solid) and three standard deviations (dotted) of all genes in each screen. (B) Individual log2 ratios of sgRNA abundance in the high and low EGFP populations for significant, overlapping genes in the 80K library (red) or 155K library (blue).

Figure 3. Validation of Genes Identified from the CRISPR/Cas Genome-wide Screen U-2 OS KO cells were transduced with AAV2-EGFP at an MOI of 12,000. Parental U-2 OS cells were used as a positive control for EGFP expression, and KIAA0319L KO cells were used a negative control for EGFP expression. 3 days post-transduction the cells were fixed and imaged. (A) Average EGFP intensity per cell was quantified. (B) Percentages of EGFP-positive cells in imaged population were quantified. Data from bar graphs depict the mean value with SD (n = 5 wells/cell line; n = 9 fields/well). (C) Representative EGFP images from the experiment. Corresponding nuclear stains (Hoechst dye, blue) are shown in the inset.

Figure 4. GPR108 and TM9SF2 Are Critical Host Cells Factors for AAV Transduction in Multiple Serotypes U-2 OS KO cells were transduced with different AAV serotypes. Parental U-2 OS cells were used as a positive control for EGFP expression, and KIAA0319L KO cells were used a negative control for EGFP expression. 3 days post-transduction the cells were fixed and imaged. The mean EGFP intensity of cells in the imaged population was quantified. Data from bar graphs depict the mean value with SD (n = 5 wells/cell line; n = 9 fields/well).

Figure 5. GPR108 Is Not Required for AAV5 Transduction (A) U-2 OS KO cells were transduced with AAV5-EGFP at an MOI of 55,000. 3 days post-transduction the cells were fixed and imaged. Representative EGFP images from the experiment are shown. Corresponding nuclear stains (Hoechst dye; blue) are shown in the inset. (B) HEP3B KO cells were transduced with AAV2-EGFP or AAV5-EGFP at an MOI of 18,000 or 55,000, respectively. 2 days post-transduction the cells were fixed and imaged. Representative EGFP images from the experiment are shown with corresponding nuclear stains in the adjacent image (AAV2 DRAQ5 stain, pseudo-color blue; AAV5, Hoechst dye). The percentage of EGFP-positive cells in the imaged population was quantified. The mean value is shown in the upper left corner (white) (AAV2: n = 2 wells/cell line; n = 9 fields/well; AAV5: n = 1 well/cell line; n = 9 fields/well). In both experiments parental cells were used as a positive control for EGFP expression, and KIAA0319L KO cells were used a negative control for EGFP expression.

Figure 6. GPR108 Predominantly Localizes to the Golgi, and GPR108 Expression Is Not Sufficient to Rescue AAV Transduction in KIAA0319L KO Cells U-2 OS parental, GPR108, and KIAA0319L KO cells expressing WT GPR108 were generated. Modified cells were transduced with AAV2-EGFP at an MOI of 12,000. 3 days post-transduction the cells were fixed and imaged. (A) Representative EGFP images from the GPR108 rescue experiment. Corresponding nuclear stains (Hoechst dye, blue) are shown in the inset. (B) The average EGFP intensity per cell was quantified from the rescue and overexpression experiments. Data from bar graphs depict the mean value with SD (n = 3 wells/cell line) (C) Left: graphical depictions of the expression cassettes used to perform the rescue and imaging experiments. Right: illustration of GPR108 with a C-terminal FLAG-tag. (D) Parental U-2 OS cells expressing the GPR108-FLAG protein were fixed and stained. Representative images depict localization of FLAG-tag GPR108 (red), Golgi (green), and Nuclei (blue). (E) HeLa cells expressing the GPR108-FLAG-GFP protein were transduced with AAV2-mCherry-capsid. After 6 h the cells were fixed and stained. Representative images depict localization…