Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Correlation between log2-fold change (L2FC) in RAD51 foci number and the Rule Set 2 on-target score. All guides targeting RAD51 regulators including RAD51 paralogs (RAD51D, RAD51C, XRCC3), BRCA1, and BRCA2 in the library are shown. Splice and nonsense variants with high Rule Set 2 score shows more significant L2FC. Pearson correlation (r) equals −0.30. (B) Quantification of RAD51 foci in irradiated S/G2-phase cells with guides targeting RAD51 regulators that have low Rule Set 2 score. Cells are grouped by sgRNA category. No. or moderate significant separation from the cells with control guides is observed. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. (Outliers are not shown in the CDFs.) (C) as in B) for guides with high on-target score, showing significant reduction in RAD51 foci in cells with nonsense and splice guides, compared to the control guides. (D) as in A) for L2FC in BRCA1 foci for BRCA1-targeting guides, showing a strong negative correlation with the Rule Set 2 score. Pearson correlation (r) equals −0.75. (E) same as B) for BRCA1 foci and guides targeting BRCA1 that have low Rule Set 2 score, showing no significant separation from the control guides. (F) same as E) for guides with high Rule Set 2 score, showing significant reduction in BRCA1 foci in cells with missense, nonsense, and splice guides. (G) Volcano plot showing no guides targeting AAVS1 and non-targeting control (NTC) guides shows statistically significant changes in RAD51 foci under irradiation. Significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.55, all AAVS1-targeting non-targeting control (NTC) guides are shown. Benjamini-Hochberg corrected two-sided KS test. (H) Same as G) showing guides that result in significant changes in RAD51 foci. (I) Gene enrichment analysis of guides targeting RAD51D among guides causing significant changes in RAD51 foci in the irradiated condition. Fisher exact test. Enriched genes are defined as p.adj <0.05. (J) same as G) for BRCA1 foci. (K) same as H) for guides that result in significant changes in BRCA1 foci (L) same as I) for BRCA1 foci.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Irradiation, Control

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Wasserstein distance of cells with DDR gene-targeting guides (Perturb) or control guides (Controls) to control cells for RAD51 foci in irradiated cells. Hits of RAD51 foci identified in the pooled screening are marked. (B) same as A) for BRCA1 foci. (C) Volcano plots showing variants yielding significant changes in the proportion of cells with high p21 expression in untreated (left) or irradiated (right) conditions. Significance is defined by p.adj < 0.05, Beta-Binomial test. The proportion of control cells with high p21 is marked with the vertical line (UNT: 0.376; IR: 0.576). sgRNA category and ClinVar category for significant guides are displayed by colors and shapes, respectively. (D) Correlation of RAD51 foci log2-fold change (L2FC) in guides delivered in the pooled library (pooled) and transduced individually (individual). The Pearson correlation (r) is 0.90. (E) same as D) for BRCA1 foci. The Pearson correlation (r) is 0.95. (F) MCF7-BE3 cells were lentivirally transduced with the DDR364 library, selected in puromycin-containing medium for 2 days, and cultured 2 days without puromycin. Cells were either untreated (UNT) or treated with DNA damaging agents Camptothecin (CPT), Olaparib (OLAP), Cisplatin (CISP) or Etoposide (ETOP) for 24 hours, then fixed before optical phenotyping and barcode detection. (G) Correlation between L2FC in RAD51 foci in CISP-treated cells and the Rule Set 2 on-target score, showing a negative correlation. All guides targeting RAD51 regulators including RAD51D, RAD51C, XRCC3, BRCA1, and BRCA2 are shown. The Pearson correlation (r) equals −0.29. (H) same as G) for OLAP-treated cells. The Pearson correlation (r) equals −0.30. (I) Volcano plot showing variants yielding significant changes in RAD51 foci in CISP-treated cells. Statistical significance is defined by p.adj < 0.05 and absolute L2FC > 0.5. All guides targeting DDR genes with on-target score >= 0.5, all AAVS1-targeting and non-targeting control (NTC) guides are shown. Two-sided KS test, *p.adj < 0.05, **p.adj < 0.01, ***p.adj < 0.001, ****p.adj < 0.0001. Same figure legend as in C). (J) same as I) for OLAP-treated cells. (K) Gene enrichment analysis in variants that result in significant changes in RAD51 foci. Fisher exact test. Enrichment is defined as p.adj <0.05. (L) Same as I) for variants yielding significant changes in large γH2AX foci in CISP-treated cells. (M) same as L) for OLAP-treated cells. (N) same as K) for large γH2AX foci.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Control, Irradiation, Expressing, Transduction, Cell Culture

Journal: bioRxiv

Article Title: CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

doi: 10.1101/2023.12.26.572587

Figure Lengend Snippet: (A) Immunoblots on guides targeting BRCA1 showing the reduction of full-length BRCA1 protein in the splice variant BRCA1.234, compared to the missense variants BRCA1.416 and.BRCA1.476, and the AAVS1 variant AAVS1.86. Cells transduced with Firefly siRNA (siFirefly) and BRCA1 siRNA (siBRCA1) were included to show the specificity of BRCA1 detection. Cells were treated with or without irradiation and the induction of DNA damage is shown with the phospho-KAP1 (pKAP1) staining. Tubulin is used as the loading control. (B) As in A) for BRCA2 variants showing the reduction of full-length BRCA2 protein in the nonsense variant BRCA2.207, compared to the missense variant BRCA2.438 and the AAVS1 variant AAVS1.86.

Article Snippet: MCF7-BE3 cells were cultured in the same medium supplemented with 2 μg/ml Blasticidin (Thermo Fisher Scientific A1113903), HT1080/Cas9 AAVS1 (Genecopoeia SL512, RRID:CVCL_C9F8) cells were cultured in the same medium supplemented with 200 μg/ml HygromycinGold B (Invivogen ant-hg-2).

Techniques: Western Blot, Variant Assay, Transduction, Irradiation, Staining, Control