Journal: bioRxiv

Article Title: The APE2 nuclease is essential for DNA double strand break repair by microhomology-mediated end-joining

doi: 10.1101/2022.07.21.500989

Figure Lengend Snippet: A. Schematic of the dual CRISPR-Cas9 screen. B. Result of the genome wide CRISPR-Cas9 screen in HT1080-BRCA1 KO compared to parental HT1080. Most significant genes are annotated. C. Result of the genome wide CRISPR-Cas9 screen in HT1080-PALB2 KO compared to parental HT1080. Most significant genes are annotated. D. Result of the dual CRISPR-Cas9 screen: Plot of MAGeCK -Log10 depletion scores of BRCA2 KO vs PALB2 KO . E. Schematic and timeline of the growth competition assay. HT1080, HT- BRCA1 KO or HT- PALB2 KO are transduced with iCas9, sgNT (non-targeting) and GFP or with RFP and the indicated sgRNA (sgGOI: gene of interest). GFP and RFP cells are mixed 1:1, treated with doxycycline (1µg/ml) to induce Cas9 expression, and subjected to flow cytometry every 2 days. F. Result of the growth competition assay at day 24. Values are % of RFP+ cells / % of GFP+ cells, normalized to day 0. 3 biological replicates. Data are mean ± s.d.

Article Snippet: For each isogenic cell line (Parental HT1080, HT1080-BRCA1 KO , and HT1080-PALB2 KO ), 150 million cells were spinfected ( ) for 2 hours at 1,000g and 33°C in 24-well plates (3.2 million cells per well in 3 ml of medium and 10 µg/mL of polybrene) with the Brunello genome-wide CRISPR/Cas9 library( ) (viral particles were directly obtained from Addgene, #179- LV73) at an MOI of 0.3.

Techniques: CRISPR, Genome Wide, Competitive Binding Assay, Transduction, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: The APE2 nuclease is essential for DNA double strand break repair by microhomology-mediated end-joining

doi: 10.1101/2022.07.21.500989

Figure Lengend Snippet: A. Schematic of wild type and mutants APE2. Amino acids mutated or deleted are indicated for both human and mouse APE2. B. Western Blot of Myc-tag (mAPE2) and Actin for the indicated MEFs used in D. C. Experimental timeline for and : On day 0, Doxycycline treatment induces expression of Cas9 ( APEX2 knockout) and mAPE2 exogenous expression. On day 3, addition of 4OHT induces TERF2 Cre recombination and telomere deprotection. On Day 8, cells are briefly treated with Colcemid and metaphase spreads are prepared. D. Quantification of telomere fusion in XRCC5 KO TERF2 F/- MEFs transduced with iCas9, sg APEX2 #1, and the indicated mAPE2 construct, and treated with doxycycline (1µg/ml) and 4OHT (1µM). EV: empty vector. 3 independent experiments. 26 metaphases were counted in each condition, each replica. Data are mean ± s.e.m. E. Western Blot of Myc-tag (hAPE2) and Actin for the indicated HT1080s used in G. F. Experimental timeline for and . Cells were treated 3 days with Doxycycline to induce expression of Cas9 ( APEX2 knockout) and of the hAPE2 exogenous constructs prior to transfection with ISce1-BFP. 24 hours post-transfection, media was replaced (with Dox). Flow cytometry was performed 6 days after ISce1 transfection. G. MMEJ quantification using reporter from 2a in HT1080- APEX2 KO clone A2.4 complemented with indicated hAPE2. mCherry+ cells (MMEJ+) were scored in the BFP+ population (I-Sce1+). Values are normalized to WT. 4 independent experiments. Data are mean ± s.e.m. Statistical analysis for D and G: One-way ANOVA. Grey: vs empty vector, Black: vs WT. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: For each isogenic cell line (Parental HT1080, HT1080-BRCA1 KO , and HT1080-PALB2 KO ), 150 million cells were spinfected ( ) for 2 hours at 1,000g and 33°C in 24-well plates (3.2 million cells per well in 3 ml of medium and 10 µg/mL of polybrene) with the Brunello genome-wide CRISPR/Cas9 library( ) (viral particles were directly obtained from Addgene, #179- LV73) at an MOI of 0.3.

Techniques: Western Blot, Expressing, Knock-Out, Transduction, Construct, Plasmid Preparation, Transfection, Flow Cytometry