Journal: bioRxiv

Article Title: CRISPR enriches for cells with mutations in a p53-related interactome, and this can be inhibited

doi: 10.1101/2021.03.10.434760

Figure Lengend Snippet: ( a ) Model describing how CRISPR and the used drugs are expected to affect targeted cells. ( b ) Growth characteristics of Hox expanded bone marrow cells (from Cas9+ GFP+ mice), exposed to CRISPR (electroporated with a GFP sgRNA, or control), or pulsed for 8h with AMG232, or Etoposide (ETOP). ( c ) Kinetic analysis of apoptosis by flow cytometry-based TUNEL assay of Hox cells exposed to CRISPR (electroporated with GFP sgRNA) or ETOP. ( d ) Kinetic qPCR analysis of Cdkn1a expression of Hox cells exposed to CRISPR (electroporated with GFP sgRNA) or ETOP. ( e ) Model describing experimental setup. ( f ) WT and Trp53 KO Hox cells (Cas9+ and GFP+) were mixed and subjected to CRISPR (electroporated with a GFP sgRNA, non-targeting ctrl (NTC) virus, CRISPR library virus, or GFP targeting sgRNA virus), or an 8h pulse with ETOP or AMG232. After seven days in culture, cells were sequenced, and the fraction of Trp53 KO sequences determined. ( g ) Hox cells (Cas9+ and GFP+) were electroporated with indicated sgRNAs, and Cdkn1a expression was analyzed by qPCR at different time points as indicated in figure. ( h ) Comparison of the Cdkn1a expression by qPCR 2h post electroporation with indicated sgRNAs, and enrichment of Trp53 KO sequences day seven. ( i ) Inhibitors used in (j-k). ( j ) WT and Trp53 KO Hox cells (Cas9+ and GFP+) were mixed and transduced with a GFP targeting sgRNA virus in the presence of inhibitors. Cells were then cultured for seven days, followed by sequencing of Trp53, and the frequency of Trp53 mutations quantified. ( k ) WT and Trp53 KO Hox cells (Cas9+ and GFP+), were mixed and transduced with a GFP targeting sgRNA virus or electroporated with a GFP sgRNA in the presence of the caspase inhibitor Z-VAD. Cells were then cultured for seven days, followed by sequencing of Trp53, and the frequency of Trp53 mutations quantified. Data is shown as mean +/− SEM, n=3 (b-d, h), mean and individual values, n=3-4 (f, j-k), or heatmap based on the average signal, n=3 (g). Data is representative of two or more experiments. n.s. = non-significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001 by two-way ANOVA and Turkey’s post-test (b-d), one-way ANOVA and Tukey’s post-test (f, j-k), or Pearson r correlation and simple linear regression line (h).

Article Snippet: Individual sgRNAs were ordered from Sigma-Aldrich, and the sgRNA library was ordered from CustomArray as a DNA oligo pool.

Techniques: CRISPR, Flow Cytometry, TUNEL Assay, Expressing, Electroporation, Transduction, Cell Culture, Sequencing

Journal: bioRxiv

Article Title: CRISPR enriches for cells with mutations in a p53-related interactome, and this can be inhibited

doi: 10.1101/2021.03.10.434760

Figure Lengend Snippet: ( a-b ) Exploratory CRISPR screen targeting 400 cell death-related genes and controls in Hox cells (a), and B16 cells (b). The sgRNA representation was analyzed by next-generation sequencing, and enrichment/depletion deconvoluted by MAGeCK. Genes (left) and individual sgRNAs of non-targeting controls (NTC), intergenic controls, Trp53, and Mdm2 (right) enriched and depleted after seven days by the CRISPR-induced DNA damage. ( c-d ) Enrichment and depletion of individual sgRNAs for Trp53 (c), and Mdm2 (d) in the CRISPR screen, comparing WT and Trp53 KO Hox and B16 cells. ( e-f ) Hox cells WT or with mutations (any Indels, including insertions and deletion of 3 nucleotides) in Mdm2 were mixed and exposed to CRISPR (sgRNA electroporation or sgRNA virus). Cells were cultured for seven days, followed by sequencing of Mdm2 to quantify the frequency of indels (e) and KO (f). ( g ) Top WP term identified by g:Profiler querying STAT1, JAK1, and PTPRC. ( h-i ) Enrichment of Jak1, Stat1, and Ptprc sgRNAs in Hox WT (h), and Hox Trp53 KO cells (i). (h) is the same data as (a), but indicating Jak1, Stat1, Ptprc. ( j ) Hox cells cultured for five days with type I interferon (IFNb) and Jak1 inhibitor. Data presented as relative cell number compared to the control (no IFNb or Jak1 inhibitor). Data is presented as volcano plots with adjusted p-values (−log10; >1.3 = p < 0.05) and Z-score (log2 fold enrichment/depletion of sgRNAs (a-b, g-h, adjusted p-value <0.05, and Z-score >1 or <−1 are indicated in color), or mean and individual values (c-f, j). * = p < 0.05, and ** = p < 0.01 by Mann-Whitney test (c, d), or one-way ANOVA and Turkey’s post-test (e-f, j).

Article Snippet: Individual sgRNAs were ordered from Sigma-Aldrich, and the sgRNA library was ordered from CustomArray as a DNA oligo pool.

Techniques: CRISPR, Next-Generation Sequencing, Electroporation, Cell Culture, Sequencing, MANN-WHITNEY

Journal: bioRxiv

Article Title: CRISPR enriches for cells with mutations in a p53-related interactome, and this can be inhibited

doi: 10.1101/2021.03.10.434760

Figure Lengend Snippet: ( a ) Model describing experimental setup. ( b-d ) Hox cells (Cas9+ and GFP+) were transduced with a custom CRISPR library and cultured for >14 days. Cells were then either exposed to CRISPR (GFP targeting sgRNA) (b), AMG232 8h pulse (c), or Etoposide 8h pulse (d). Cells were cultured for seven days and then the sgRNA representation was analyzed by next-generation sequencing, and enrichment/depletion deconvoluted by MAGeCK. ( e-h ) Z-scores of individual sgRNAs (n=4/gene) for Atm (e), Chek2 (f), Trp53 (g), and Cdkn1a (h) in WT and Trp53 KO Hox cells treated with mock (electroporation without sgRNA), CRISPR (electroporation with GFP targeting sgRNA), AMG232 or ETOP. ( i ) Z-scores of individual sgRNAs (n=4/gene) in Hox cells treated with Trp53 siRNA at the same time as being electroporated with a GFP targeting sgRNA as described in (a). ( j ) Z-scores of individual sgRNAs (n=4/gene) for genes linked to apoptosis. ( k ) Model indicating genes playing a non-redundant role in the DNA damage response. Data presented as volcano plots with Z-score (log2 fold change) and adjusted p-values (b-d), or mean and individual values for 4 gRNAs from the exploratory screen (e-j). * = P < 0.05, and n.s. = non-significant by Mann-Whitney test.

Article Snippet: Individual sgRNAs were ordered from Sigma-Aldrich, and the sgRNA library was ordered from CustomArray as a DNA oligo pool.

Techniques: Transduction, CRISPR, Cell Culture, Next-Generation Sequencing, Electroporation, MANN-WHITNEY

Journal: bioRxiv

Article Title: CRISPR enriches for cells with mutations in a p53-related interactome, and this can be inhibited

doi: 10.1101/2021.03.10.434760

Figure Lengend Snippet: ( a ) Enrichment score of TP53 gRNAs in 808 cell lines, stratified based on the presence or absence of any mutation in TP53. ( b ) Correlation between the enrichment of TP53 sgRNAs and AMG232 sensitivity. TP53 WT cells are indicated in red. ( c ) Sensitivity to AMG232 in cell lines stratified based on TP53 sgRNA enrichment. ( d ) Correlation between the enrichment of TP53 sgRNAs and MDM2 sgRNAs. ( e ) Top 10 genes with the strongest positive (+) and negative (−) correlation with TP53 sgRNA enrichment from full genome CRISPR screens of 808 cell lines. * indicates genes identified as transcription factor target genes for p53 identified by Enricher. Bold indicates genes identified experimentally in figure 2. ( f ) Physical interactions of genes in (e) defined by geneMANIA. + indicates genes that positively correlate, and - indicates genes that negatively correlate with TP53 sgRNA enrichment. ( g ) Correlation of TP53 sgRNA enrichment to baseline CDKN1A expression. TP53 WT cells are indicated in red. ( h ) Baseline CDKN1A expression in cells stratified based on the enrichment of TP53 sgRNAs. ( i ) Top 10 genes with expression correlating with enrichment of TP53 sgRNAs. * indicates genes identified as transcription factor target genes for p53. ( j ) tSNE dimensionality reduction analysis of cell, n=808, based on expression of the ten genes in (i). Data includes all available data in the Depmap CRISPR (Avana) 20Q4, Expression Public 20Q4, as well as Drug sensitivity (PRISM Repurposing Primary Screen) 19Q4 releases. Each dot represents one cell line (a-d, g, h, j), and the data is based on n=808 (a, d-f) n=408 (b-c), n=800 (g-j). Statistic based on unpaired T-test (a, c, h), Pearson r correlation and simple linear regression line (d), and calculated in Depmap (e, i).

Article Snippet: Individual sgRNAs were ordered from Sigma-Aldrich, and the sgRNA library was ordered from CustomArray as a DNA oligo pool.

Techniques: Mutagenesis, CRISPR, Expressing

Journal: Nature protocols

Article Title: Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning

doi: 10.1038/s41596-021-00669-0

Figure Lengend Snippet: Troubleshooting table

Article Snippet: To identify an sgRNA target sequence using the GenScript sgRNA database ( https://www.genscript.com/gRNA-database.html ) or other webtools, input the gene name, symbol or ID and search for human SpCas9 gRNA sequences.

Techniques: Negative Control, Ligation, Transformation Assay, Plasmid Preparation, Selection, Passaging, Clone Assay, Concentration Assay, Sequencing