grna sequence for tp53  (Addgene inc)

Journal: Genome Research

Article Title: APOBEC3A drives deaminase mutagenesis in human gastric epithelium

doi: 10.1101/gr.280338.124

Figure Lengend Snippet: Mutational impact of APOBEC-associated single base substitutions in DNA and RNA. ( A ) Mutational burden of SNVs following APOBEC overexpression (A3A or A3B), measured by whole-genome sequencing of the clones and duplex DNA sequencing. The number of SNVs measured by the duplex DNA sequencing was normalized per diploid genome; ( left ) hGO iA3A lines, and ( right ) hGO iA3B lines. ( B ) Mutational burden of APOBEC-associated SNVs in the hGO iA3A and TP53 KO-hGO iA3A clone sequencing. The number of A3A-associated SNVs (SBS2+SBS13) in hGO iA3A and TP53 KO-hGO iA3A clones under each condition. Statistical significance was determined using a t -test: (*) P < 0.05, (n.s.) not significant. ( C ) Number of A3A-associated SNVs (SBS2+SBS13; normalized per diploid genome) in BotSeqS results for hGO iA3A lines under each doxycycline treatment condition. Black lines represent 95% confidence intervals based on a Poisson distribution. ( D ) Mutational burden of APOBEC-associated SNVs in the hGO iA3B and TP53 KO-hGO iA3B clone sequencing; the number of A3B-associated SNVs (SBS2+SBS13) in hGO iA3B and TP53 KO-hGO iA3B clones under each condition. Statistical significance was determined using a t -test. ( E ) Number of A3B-associated SNVs (SBS2+SBS13; normalized per diploid genome) in BotSeqS results for hGO iA3B lines under each doxycycline treatment condition. Black lines represent 95% confidence intervals based on a Poisson distribution. ( F ) Mutational burden and spectra of APOBEC-associated SNVs in each experimental condition. The number of SNVs in BotSeqS results were normalized per diploid genome; ( left ) hGO iA3A lines, and ( right ) hGO iA3B lines. ( G ) Number of C > U RNA editing in bulk RNA-seq in hGO iA3A lines (n = 3 per condition), normalized per 3.1 Gb of mapped bases. ( H ) Spectra of RNA editing in trinucleotide contexts in hGO iA3A lines. ( I ) Number of C > U RNA editing in bulk RNA-seq in hGO iA3A lines (n = 3 per condition), normalized per 3.1 Gb of mapped bases. ( J ) Spectra of RNA editing in trinucleotide contexts in hGO iA3B lines.

Article Snippet: Two vectors were purchased: (1) CMV-rtTA-Hyg R vector (Addgene, #102423) and (2) CRISPR- Cas9 vectors containing gRNA sequence for TP53 (Addgene, #121917).

Techniques: Over Expression, Sequencing, Clone Assay, DNA Sequencing, RNA Sequencing

Journal: Genome Research

Article Title: APOBEC3A drives deaminase mutagenesis in human gastric epithelium

doi: 10.1101/gr.280338.124

Figure Lengend Snippet: Characteristics of A3A-associated mutational signatures. ( A ) Context preference of A3A between YpTp C pA and RpTp C pA context in hGO iA3A lines and APOBEC-associated mutations in hypermutant cancer samples. Only PCAWG cancer samples with a combined APOBEC-associated clonal mutational burden (SBS2+SBS13) greater than 5000 were selected (n = 63) among eight cancer types with a high prevalence of APOBEC mutational activity: lung adenocarcinoma (n = 15), breast adenocarcinoma (n = 12), bladder urothelial carcinoma (n = 11), head-and-neck squamous cell carcinoma (n = 13), lung adenocarcinoma (n = 6), uterine corpus endometrial carcinoma (n = 3), esophageal adenocarcinoma (n = 2), and stomach adenocarcinoma (n = 1). Dashed black line, expected; orange line, hGO iA3A ; dark brown line, cancer. ( B ) Correlation between A3A-associated base substitutions and ID9 contributing indels among hGO iA3A lines. ( C ) Associations between A3A-associated (SBS2 and SBS13) and age-associated (SBS5 and SBS40) SNVs among hGO iA3A lines and TP53 KO-hGO iA3A clones. ( D ) Changes in POLH gene expression (translesion synthesis DNA polymerase) following A3A induction in hGO iA3A and TP53 KO-hGO iA3A lines.

Article Snippet: Two vectors were purchased: (1) CMV-rtTA-Hyg R vector (Addgene, #102423) and (2) CRISPR- Cas9 vectors containing gRNA sequence for TP53 (Addgene, #121917).

Techniques: Activity Assay, Clone Assay, Gene Expression, Translesion Synthesis

Journal: Genome Research

Article Title: APOBEC3A drives deaminase mutagenesis in human gastric epithelium

doi: 10.1101/gr.280338.124

Figure Lengend Snippet: Genomic and epigenomic distribution of APOBEC3A-associated mutations. ( A ) Correlations between epigenetic markers and A3A-associated substitutions. ( B ) Fold change of mutation rates of A3A-associated SNVs across genomic regions grouped by replication timing. Data are presented as mean ± 95% confidence interval. ( C ) Mutation rates on the leading and lagging DNA strands during replication. Statistical significance was determined using a χ 2 test: (****) P < 0.00005. ( D ) Fold change of mutation rates of A3A-associated SNVs across genomic regions grouped by transcription. Data are presented as mean ± 95% confidence interval. ( E ) Mutation rates on the transcribed and untranscribed DNA strands during transcription. Statistical significance was determined using a χ 2 test: (***) P < 0.0005. ( F ) Mutation rates across subgenic regions (5′-UTR, introns, protein coding sequences [CDSs], and 3′-UTR) in hGO iA3A clones ( left ) and TP53 KO-hGO iA3A clones ( right ). Red dashed line, average genome-wide mutation rate.

Article Snippet: Two vectors were purchased: (1) CMV-rtTA-Hyg R vector (Addgene, #102423) and (2) CRISPR- Cas9 vectors containing gRNA sequence for TP53 (Addgene, #121917).

Techniques: Mutagenesis, Clone Assay, Genome Wide

Journal: eLife

Article Title: Plectin-mediated cytoskeletal crosstalk as a target for inhibition of hepatocellular carcinoma growth and metastasis

doi: 10.7554/eLife.102205

Figure Lengend Snippet: ( A ) Hepatocellular carcinoma (HCC)-predisposing lesions were introduced by hydrodynamic gene delivery via tail vein injection (HDTVi) of transposon vector encoding MYC in conjunction with CRISPR/Cas9 construct targeting Tp53 (Myc;sgTp53) in Plec fl/fl and Plec ΔAlb cohorts of 7-wk-old male mice. Plec fl/fl mice were kept either untreated or every second day provided with orogastric gavage of plecstatin ( Plec fl/fl +PST) and the development of HCC was monitored by MRI at 11, 13, and 16 wk, as indicated. Representative MRI images of Plec fl/fl and Plec ΔAlb and Plec fl/fl +PST tumors acquired at indicated time points. Dashed circles, tumors. Scale bar, 2 cm. Graphs show the average number of tumors (lower graph) and percentual tumor incidence (upper graph) inferred from MRI images. N=5 ( Plec fl/fl ), 7 ( Plec ΔAlb), 4 ( Plec fl/fl +PST). ( B ) Myc;sgTp53 HCC was induced in Plec fl/fl , Plec ΔAlb, and PST-treated Plec fl/fl ( Plec fl/fl +PST) male mice as in ( A ). Shown are representative images of Plec fl/fl , Plec ΔAlb, and Plec fl/fl +PST livers from mice with fully developed multifocal HCC sacrificed 6 wk post-induction. Scale bar, 1 cm. Boxplots show tumor burden in the livers expressed as the liver/body weight ratio (upper graph) and number of tumors per mouse (lower graph). The box represents the median, 25 th , and 75 th percentile with whiskers reaching the last data point; dots, mice; N=12 ( Plec fl/fl ), 9 ( Plec ΔAlb), 10 ( Plec fl/fl +PST). Two-tailed t -test; *p<0.05. ( C ) Representative images of H&E-stained Plec fl/fl , Plec ΔAlb, and Plec fl/fl +PST liver sections. Note darker areas corresponding to HCC lesions. Boxed areas, 12x images. Scale bars, 5 and 1 mm (boxed areas). ( D ) Gene set enrichment analysis of differentially regulated proteins in Plec fl/fl vs Plec ΔAlb and Plec fl/fl vs Plec fl/fl +PST livers from the cohort shown in ( A ). Prediction of canonical signaling pathways in Plec fl/fl vs Plec ΔAlb (left) and Plec fl/fl vs Plec fl/fl +PST (right) proteomes. ( E ) Association of plectin-dependent signatures compiled from human HCC-derived cells (see ) and mouse models (see D ) with plectin ( PLEC ) mRNA expression in HCC patients. Right panel shows the levels of selected signatures in patients grouped into quartiles of PLEC expression level. N=1268. p-values were generated from an analysis of variance (ANOVA).

Article Snippet: For hydrodynamic tail vein injections, a mixture of a plasmid mix containing 5 μg/ml of px330 expressing Tp53 sgRNA, 5 μg/ml of pT3-EF1a MYC DNA (92046, Addgene, Watertown, MA, USA), and 0.5 μg/ml pCMV HSB2 sleeping beauty transponase was prepared in a sterile 0.9% sodium chloride (NaCl) solution.

Techniques: Injection, Plasmid Preparation, CRISPR, Construct, Two Tailed Test, Staining, Protein-Protein interactions, Derivative Assay, Expressing, Generated

Journal: Cancer Discovery

Article Title: Disparate Pathways for Extrachromosomal DNA Biogenesis and Genomic DNA Repair

doi: 10.1158/2159-8290.CD-23-1117

Figure Lengend Snippet: The impact of 3D chromosome conformation on ecDNA formation. Schematic depictions of ( A ) the workflow for examining the impact of chromosome conformation on circularization efficiency and ( B ) the chromosome 1 loop, with the approximate positions of the CRISPR-C guide RNAs used. Hap1 Hi-C interaction maps of ( C ) an approximately 1-Mb loop on chromosome 1 and ( D ) an approximately 800-kb loop on chromosome 6. Gray arches, loops; black lines, guide locations. E, ecDNA frequency generated by indicated chr1 guide pair. Line, mean. F, ecDNA frequency after normalization for individual guide editing efficiency ( n = 3; error bars = SEM). G, ecDNA frequency generated by indicated chr6 guide pair. Line, mean. H, ecDNA frequency after normalization for individual guide editing efficiency. Dot, mean ( n = 3; error bars = SEM). P values in F and H were calculated with unpaired two-sided t tests. ecDNA and scar junction frequencies at 24 hours in ( I ) Hap1 cells, ( J ) K562 cells, or ( K ) FTE5 (TP53 −/− ) organoids. P values were calculated with paired two-sided t tests. Bars, means ( n = 3 biological replicates; error bars = SEM). MYCe2, MYC ecDNA 2. ( B, Created with BioRender.com .)

Article Snippet: TP53 was knocked out of FTE5 by dissociating organoids to a single-cell suspension as above and electroporating in a PX330 plasmid transiently expressing both Cas9 and a gRNA targeting exon 4 of TP53 (Addgene, #121917) using a Lonza 4D-Nucleofector X (#AAF-1003X) with protocol Primary Cell P3 code CA137.

Techniques: CRISPR, Hi-C, Generated