Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications

doi: 10.1038/s41374-019-0283-0

Figure Lengend Snippet: A schematic overview of the Cas9 enrichment workflow showing the wet laboratory (A) and informatics steps (B) . Cleavage reactions for (+) and (−) strand guide RNAs are performed separately to prevent interference.

Article Snippet: For each locus, PAM sites for two custom Alt-R ® CRISPR-Cas9 guide RNAs, designed using an online tool ( https://eu.idtdna.com/ ), were targeted to linearize the genomic DNA on either the ‘+ or ‘−‘ strand (design IDs and sequences are listed in Supplementary Table 1 ).

Techniques:

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications

doi: 10.1038/s41374-019-0283-0

Figure Lengend Snippet: Long-read analysis of the VHL locus (Case 1). (A) Reads originating from guide RNA CD.Cas9.XWFV6878.AC and (B) reads originating from guide RNA CD.Cas9.XWFV6878.AA. Each read alignment was split into its 5′ and 3′ component; these data can be reconciled using the displayed read ID. MinION reads mapping to the (+) strand are colored gray, and those mapping to the (−) strand are green. For each Illumina read-pair, the read 1 alignment is colored purple and the read 2 alignment is turquoise. ^ denotes a read’s start site. * denotes a read’s end position. Genomic coordinates refer to human reference sequence build hg19.

Article Snippet: For each locus, PAM sites for two custom Alt-R ® CRISPR-Cas9 guide RNAs, designed using an online tool ( https://eu.idtdna.com/ ), were targeted to linearize the genomic DNA on either the ‘+ or ‘−‘ strand (design IDs and sequences are listed in Supplementary Table 1 ).

Techniques: Sequencing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications

doi: 10.1038/s41374-019-0283-0

Figure Lengend Snippet: Long-read analysis of the DMD locus (Case 2). (A) Reads originating from guide RNA CD.Cas9.MGPL4222.AA and (B) reads originating from guide RNA CD.Cas9.MFWR5781.AA. Each read alignment was split into its 5′ and 3′ component; these data can be reconciled using the displayed read ID. MinION reads mapping to the (+) strand are colored grey, and those mapping to the (−) strand are green. ^ denotes a read’s start site. * denotes a read’s end position. Genomic coordinates refer to human reference sequence build hg19.

Article Snippet: For each locus, PAM sites for two custom Alt-R ® CRISPR-Cas9 guide RNAs, designed using an online tool ( https://eu.idtdna.com/ ), were targeted to linearize the genomic DNA on either the ‘+ or ‘−‘ strand (design IDs and sequences are listed in Supplementary Table 1 ).

Techniques: Sequencing

Journal: STAR Protocols

Article Title: CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells

doi: 10.1016/j.xpro.2021.100407

Figure Lengend Snippet: DNA-PKcs inhibition augments the efficiency of HDR-mediated gene editing Homozygous tagging of the centriolar distal appendage protein SCLT1 with the V5-epitope is presented as example. Characterization of the polyclonal population of RPE1 cells resulting from the electroporation with a SCLT1-V5-tagging RNP mixture (Cas9, SCLT1 gRNA and SCLT1-V5 Ultramer®) in the absence (DMSO) or presence (NU7441) of the DNA-PKcs inhibitor. (A) Centrioles are stained with anti-γTub antibody, V5 signal (if present) co-localizes with one of the two γTub-positive centrioles. White arrows indicate V5+ cells. Scale bar, 10 μm. Blow-ups without Hoechst 33342 are magnified 2.5×. (B) Bar chart showing the percentage of V5+ cells for each condition. N=3 independent experiments (with >100 cells visually scored for each condition), unpaired Student’s t test, ∗∗ = p < 0.05. (C) Electroporated RPE1, both untreated (DMSO) and treated with NU7441 were probed for V5 and compared to RPE1 parental cells. V5 signal, when present, corresponds to the expected molecular weight of 75 kDa.

Article Snippet: IDT™ Custom Alt-R® CRISPR-Cas9 guide RNA , N/A , https://eu.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM.

Techniques: Inhibition, Electroporation, Staining, Molecular Weight

Journal: STAR Protocols

Article Title: CRISPR/Cas9 ribonucleoprotein-mediated knockin generation in hTERT-RPE1 cells

doi: 10.1016/j.xpro.2021.100407

Figure Lengend Snippet:

Article Snippet: IDT™ Custom Alt-R® CRISPR-Cas9 guide RNA , N/A , https://eu.idtdna.com/site/order/designtool/index/CRISPR_CUSTOM.

Techniques: Recombinant, Purification, Plasmid Preparation, CRISPR, Electroporation, Software

Journal: NPJ Precision Oncology

Article Title: Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer

doi: 10.1038/s41698-025-00837-5

Figure Lengend Snippet: A Overview for establishing an hiPSC model of breast cancer: primary breast tumor cells are dissociated and reprogrammed into hiPSCs (BC-hiPSCs), followed by differentiation into mammary epithelial cells (BC-hiPSC-MECs) for drug response phenotyping. B Number of BC-hiPSC colonies generated following different reprogramming methodologies, where each row is an independent experiment (OSKM = OCT4 /SOX2/ KLF4 / MYC expression; M87 = breast tumor cell media; B8T/B8 = hiPSC media; n = 2). C Representative phase-contrast images of primary breast cancer cells during reprogramming showing emergence of BC-hiPSC colony. D Flow cytometry assessment of TRA-1-60 and SSEA4 expression in control and BC-hiPSC lines ( n = 3–5). E Flow cytometry assessment of TNNT2 expression in control and BC-hiPSC lines differentiated into cardiomyocytes ( n = 3–6). F Flow cytometry assessment of HNF4A expression in control and BC-hiPSC lines differentiated into hepatocytes ( n = 3–7). G Flow cytometry assessment of NES expression in control and BC-hiPSC lines differentiated into neural progenitor cells ( n = 2-4). H Sanger sequencing of BC-hiPSCs for patient-specific variants in AXIN2 (BC3), BRCA2 (BC6), and BRCA1 (BC8). Variant allele frequency for variants in BC-hiPSCs and isogenic healthy cells from BC6 ( I ) and BC8 ( J ) patients determined by whole genome sequencing. n = experimental replicates, ANOVA, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005, **** P ≤ 0.0001, ns = not significant. Scale bars represent 100 µm.

Article Snippet: Custom, guide-specific CRISPR RNA (crRNA) oligonucleotides targeting BRCA1 were designed using Benchling and CRISPOR design tools (see Supplementary Data for crRNA sequences). crRNA (IDT) and tracrRNA (IDT) were reconstituted in nuclease free duplex buffer (IDT, 11-01-03-01) to a concentration of 200 μM. crRNA and tracrRNA were combined 1:1 (final duplex concentration of 100 μM) and heated at 95 °C for 5 min. After allowing it to cool to RT, the duplex was combined with Alt-R S.p.

Techniques: Generated, Expressing, Flow Cytometry, Control, Sequencing, Variant Assay

Journal: NPJ Precision Oncology

Article Title: Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer

doi: 10.1038/s41698-025-00837-5

Figure Lengend Snippet: A Sanger sequencing of unedited isogenic (ISO) and BRCA1 knockout (KO) hiPSC lines. gRNA target and PAM site indicated. B BRCA1 expression in ISO and BRCA1 KO hiPSC lines by RT-PCR, normalized to ISO ( n = 3). Effect of olaparib treatment (6 days) on ISO and BRCA1 KO hiPSC-MEC viability indicated by resazurin-based kill curve ( C ) and LD 50 ( D ) ( n = 5). Effect of doxorubicin treatment (3 days) on ISO and BRCA1 KO hiPSC-MEC viability indicated by resazurin-based kill curve ( E ) and LD 50 ( F ) ( n = 5). Quantification of γH2AX ( G ) and RAD51 ( H ) staining intensity in ISO and BRCA1 KO hiPSC-MECs treated with indicated concentration of olaparib or DMSO (0 μM), normalized to 0 μM ( n ≥ 74 cells/condition). LD 50 values (mean and SEM) are presented in Supplementary Table . n = experimental replicates, unpaired Student’s T -test, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005, **** P ≤ 0.0001, ns not significant.

Article Snippet: Custom, guide-specific CRISPR RNA (crRNA) oligonucleotides targeting BRCA1 were designed using Benchling and CRISPOR design tools (see Supplementary Data for crRNA sequences). crRNA (IDT) and tracrRNA (IDT) were reconstituted in nuclease free duplex buffer (IDT, 11-01-03-01) to a concentration of 200 μM. crRNA and tracrRNA were combined 1:1 (final duplex concentration of 100 μM) and heated at 95 °C for 5 min. After allowing it to cool to RT, the duplex was combined with Alt-R S.p.

Techniques: Sequencing, Knock-Out, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Concentration Assay

Journal: NPJ Precision Oncology

Article Title: Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer

doi: 10.1038/s41698-025-00837-5

Figure Lengend Snippet: A Sanger sequencing of unedited isogenic control (ISO) hiPSC line and hiPSC line with BRCA1 c.68_69delAG variant introduced (INTRO). gRNA target, PAM site, and silent mutation (*) indicated. B Sanger sequencing of unedited BC8-1 hiPSC line and BC8-1 hiPSC line with BRCA1 c.68_69delAG variant corrected (CORR). gRNA target, PAM site, and silent mutations (*) indicated. Effect of olaparib treatment (6 days) on ISO, INTRO, BC8-1, and CORR hiPSC-MEC viability indicated by resazurin-based kill curve ( C ) and LD 50 ( D ) ( n = 5–6). Effect of doxorubicin treatment (3 days) on ISO, INTRO, BC8-1, and CORR hiPSC-MEC viability indicated by resazurin-based kill curve ( E ) and LD 50 ( F ) ( n = 5-6). Quantification of γH2AX ( G ) and RAD51 ( H ) staining intensity in ISO and INTRO hiPSC-MECs treated with indicated concentration of olaparib or DMSO (0 μM), normalized to 0 μM ( n ≥ 174 cells/condition). Quantification of γH2AX ( I ) and RAD51 ( J ) staining intensity in BC8-1 and CORR hiPSC-MECs treated with indicated concentration of olaparib or DMSO (0 μM), normalized to 0 μM ( n ≥ 140 cells/condition). LD 50 values (mean and SEM) are presented in Supplementary Table . n = experimental replicates, unpaired Student’s T -test, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005, **** P ≤ 0.0001, ns not significant.

Article Snippet: Custom, guide-specific CRISPR RNA (crRNA) oligonucleotides targeting BRCA1 were designed using Benchling and CRISPOR design tools (see Supplementary Data for crRNA sequences). crRNA (IDT) and tracrRNA (IDT) were reconstituted in nuclease free duplex buffer (IDT, 11-01-03-01) to a concentration of 200 μM. crRNA and tracrRNA were combined 1:1 (final duplex concentration of 100 μM) and heated at 95 °C for 5 min. After allowing it to cool to RT, the duplex was combined with Alt-R S.p.

Techniques: Sequencing, Control, Variant Assay, Mutagenesis, Staining, Concentration Assay

Journal: NPJ Precision Oncology

Article Title: Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer

doi: 10.1038/s41698-025-00837-5

Figure Lengend Snippet: Summary of breast cancer patient recruitment and BC-hiPSC generation

Article Snippet: Custom, guide-specific CRISPR RNA (crRNA) oligonucleotides targeting BRCA1 were designed using Benchling and CRISPOR design tools (see Supplementary Data for crRNA sequences). crRNA (IDT) and tracrRNA (IDT) were reconstituted in nuclease free duplex buffer (IDT, 11-01-03-01) to a concentration of 200 μM. crRNA and tracrRNA were combined 1:1 (final duplex concentration of 100 μM) and heated at 95 °C for 5 min. After allowing it to cool to RT, the duplex was combined with Alt-R S.p.

Techniques:

Journal: bioRxiv

Article Title: TECPR1 provides E3-ligase like activity to the ATG5-ATG12 complex to conjugate LC3/ATG8 to damaged lysosomes

doi: 10.1101/2023.06.24.546289

Figure Lengend Snippet: Panel A. Domain map of TECPR-1 showing site of insertion of stop codon by custom CRISPR gRNA. Atg16L1-/-MEFs and gene edited Atg16L1-/-MEFS expressing truncated TECPR1* were incubated in nutrient media with chloroquine (100mM, panel B ) or LLOMes (1mM panel C ). Cells were fixed and immunostained for LC3(green) and galectin-3 (red). Rendered images (v and vi, xi and xii) were used to count puncta. Higher magnification images of boxed ROI are shown in iv and x. The graphs compare numbers of LC3 puncta co-localised with galectin-3 counted from rendered images of 20 cells incubated with chloroquine, P-values were calculated using multiple t-test (***P < 0.001). ( panel D ) or LLOMes ( panel F ). Generation of LC3II was assessed by western blot for LC3 (chloroquine panel E ), (LLOMes panel G ).

Article Snippet: Gene editing of Tecpr1 in MEFs was achieved using custom CRISPR gRNA lentivirus transduction particles (Mission, Sigma Aldrich).

Techniques: CRISPR, Expressing, Incubation, Western Blot