Journal: International Journal of Molecular Sciences

Article Title: The Role of Trio, a Rho Guanine Nucleotide Exchange Factor, in Glomerular Podocytes

doi: 10.3390/ijms19020479

Figure Lengend Snippet: Trio Contributes to Basal Rac1 Activity and Cell Size ( a ) The CRISPR/Cas9 system was used to create Trio KO human podocytes. Monoclonal populations were selected and tested for Trio expression using immunoblotting. Although the KO was not complete, it decreased Trio expression by 60% (Trio KO #13) or 58% (Trio KO #14) when normalized to calnexin and then to control Cas9. ECL was used for visualization; ( b , c ) Cdc42/Rac interactive binding (CRIB) pulldown (PD) assays revealed that both Trio KO #13 (0.39 ± 0.10, n = 3, ** p < 0.01 compared to control Cas9) and Trio KO #14 (0.24 ± 0.06, n = 3, *** p < 0.001 vs. control Cas9) had decreased Rac1 activity compared to control Cas9. ECL was used for visualization and quantification was done using densitometric analysis. Results were normalized to tubulin and then to control Cas9 cells; ( d ) MTT assay showed that the proliferation rate of Trio KOs did not differ from that of control Cas9 cells between 24 h and 48 h after plating ( y -axis represents the value at 48 h over the value at 24 h). Trio KOs #9 and #19 also had decreased Trio expression (see ) and were used for the MTT assay; ( e , f ) Control Cas9 and Trio KOs were fixed and stained for phalloidin. Trio KOs (2349 ± 114 μm 2 , n = 89 cells from two experiments, *** p < 0.001 vs. control Cas9) were significantly smaller than control Cas9 (3167 ± 140 μm 2 , n = 81 from two experiments). Scale bar in Cas9 photo ( e ) is 10 μm and is the same for Trio KO photo ( g , h ) GFP-alone or GFP-Trio-GEFD1 were expressed in HP and cells were stained for phalloidin. GFP-Trio-GEFD1 expressing cells were larger (3223 ± 131 μm 2 , n = 65 from two experiments, *** p < 0.001 vs. GFP-alone) than GFP-alone expressing cells (2327 ± 142 μm 2 , n = 61 from two experiments). Scale bar in ( g ) is 10 μm.

Article Snippet: The lentiviral Cas9 and sgRNA Trio were from Genecopoeia (Thorold, ON, Canada).

Techniques: Activity Assay, CRISPR, Expressing, Western Blot, Control, Binding Assay, MTT Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Role of Trio, a Rho Guanine Nucleotide Exchange Factor, in Glomerular Podocytes

doi: 10.3390/ijms19020479

Figure Lengend Snippet: Trio affects motility, attachment, and vinculin distribution. ( a ) Control Cas9 and Trio KOs were allowed to grow to confluency, serum starved overnight, and then a scratch wound was created. Images were taken at time 0 and 5 h after; ( b ) Cell motility was calculated from percent wound closure from 0 to 5 h. Control Cas9 cells had a greater wound closure than Trio KOs (control Cas9: 21.4 ± 0.55, Trio KOs: 17.3 ± 0.99, n = 4, * p < 0.05 vs. control Cas9). The yellow lines have been drawn to easily visualize the two sides of the wound. Scale bar in ( a ) is 100 μm; ( c ) The same number of control Cas9 and Trio KOs were plated on laminin 521 and allowed to attach for 1.25 h. Attached cells were quantified by crystal violet as described in Methods. Control Cas9 cells had more attachment than Trio KOs (absorbance values at 550 nm: control Cas9: 0.17 ± 0.01, Trio KO: 0.13 ± 0.01, n = 3–4, * p < 0.05 vs. control Cas9); ( d ) Control Cas9 and Trio KOs were plated overnight on laminin 521, fixed, and stained for phalloidin (red) and vinculin (green). Co-localization is shown in yellow in merged photo. Scale bar in ( d ) is 10 μm. The amount of vinculin at the periphery was quantified; ( e ) Quantification of peripheral vs. central vinculin staining from ( e ) was performed as described in Methods. Trio KOs had more peripheral vinculin than control Cas9 (control Cas9: 0.55 ± 0.02, n = 21 cells counted from two experiments; Trio KO: 0.72 ± 0.04, n = 19 cells counted from two experiments, *** p < 0.001 vs. control Cas9).

Article Snippet: The lentiviral Cas9 and sgRNA Trio were from Genecopoeia (Thorold, ON, Canada).

Techniques: Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Role of Trio, a Rho Guanine Nucleotide Exchange Factor, in Glomerular Podocytes

doi: 10.3390/ijms19020479

Figure Lengend Snippet: TGFβ1 decreases Rac1 activity in Trio KOs, while increasing CdGAP activation in control cells. ( a ) Control Cas9 and Trio KOs were incubated with TGFβ1 (10 ng/mL) for 60 min. Cell lysates were subjected to CRIB PD and immunoblotting for Rac1 and tubulin. ECL was used for visualization; ( b ) Densitometric analysis. Results were first normalized to tubulin and then fold increase of +TGFβ1 over −TGFβ1 was calculated. In control Cas9 cells, TGFβ1 increased Rac1 activity 1.72-fold ± 0.20, n = 4; whereas in Trio KOs, TGFβ1 decreased Rac1 activity 0.59-fold ± 0.13, n = 4. ** p < 0.01 vs. control Cas9; ( c ) HP were treated with TGFβ1 for the indicated times and cell lysates were pulled down with GST-CA-Rac1. Precipitates and lysates were immunoblotted for CdGAP and tubulin and visualized using LiCor; ( d ) Densitometric analysis. Levels of active CdGAP were normalized to CdGAP levels in the total lysate (i.e., input) and then to 0 min. CdGAP activity was increased at 30 min (1.55-fold ± 0.16, * p < 0.05 vs. unstimulated), 45 min (1.56-fold ± 0.21, * p < 0.01 vs. unstimulated), and 60 min (1.53-fold ± 0.12, ** p < 0.05 vs. unstimulated), n = 3–4; ( e ) Control Cas9 cells and Trio KOs were stimulated with TGFβ1 (10 ng/mL, 60 min), cell lysates were immunoblotted for p-p38 and total p38, and protein was visualized using ECL; ( f ) Densitometric analysis. Basal (unstimulated) levels of p-p38 were normalized to tubulin and then to control Cas9 levels. Trio KOs had 0.55 ± 0.28 fold decrease, n = 4, * p < 0.05 versus control Cas9; ( g ) Results were normalized to tubulin and then fold increase of +TGFβ1 over −TGFβ1 was calculated. TGFβ1 increased p-p38 more in Trio KOs than in control Cas9 (control Cas9: 1.54 ± 0.45 fold, Trio KO: 2.71 ± 0.70 fold, n = 4, * p < 0.05 vs. control Cas9).

Article Snippet: The lentiviral Cas9 and sgRNA Trio were from Genecopoeia (Thorold, ON, Canada).

Techniques: Activity Assay, Activation Assay, Control, Incubation, Western Blot

Journal: bioRxiv

Article Title: In vivo CRISPR screens identify key modifiers of CAR T cell function in myeloma

doi: 10.1101/2024.11.19.624352

Figure Lengend Snippet: a. Composition of genes targeted in the Mario library based on gene function. b. Diagram of Mario-CAR T cell production and screen workflow. T cells were activated with anti-CD3/CD28 microbeads, transduced one day later sequentially with the BCMA CAR and sgRNA library lentiviruses. A pre-electroporation sample was frozen down for analysis on day –11 (Baseline), and the remaining cells underwent Cas9 mRNA electroporation (day –7) and CD3 negative selection (day –5). Transduction efficiencies were assessed on day –4 and 0 at the end of production prior to cryopreservation. Mario-CAR T cells (2E6 double positive cells) were transferred into NSG mice bearing MM.1S multiple myeloma. At day 7 (early in vivo) or day 21 (late in vivo), mice were euthanized and a total marrow harvest was conducted (collecting femur, tibia, and spine) for isolation of Mario-CAR T cells. Mario-CAR T cells were produced from n=3 healthy donor T cells in IL-2 (ND216, ND99, ND106) and n=2 of the same donors for IL7/15 (ND106, ND216). c. Genes ranked by Log2(fold change) during in vitro manufacturing (end-of-production vs. baseline, left panel) and early in vivo (day 7 vs. end-of-production, middle panel) or late in vivo (day 21 vs. end-of-production, right panel). Enriched genes are shown in red and depleted genes are shown in blue with circle size corresponding to –log10 (FDR). n = 3 healthy donor T cells (ND216, ND99, ND106). d. Frequency histograms of enrichment or depletion of sgRNAs for RASA2 , PTPN2 , and CDKN1B , grouped by respective period. n = 3 healthy donor T cells (ND216, ND99, ND106). e. LFC comparison for gene knockout scoring between Mario-CAR T cells produced in IL-2 vs. IL-7/IL-15 at individual time points. n = 3 healthy donor T cells for IL2 (ND216, ND99, ND106) and 2 of the 3 donors for IL7/15 (ND106, ND216). f. Abundance of sgRNAs targeting individual genes across the entire screen workflow for Mario-CAR T cells produced in IL-2. b was created with Biorender.com. ND: normal donor. LFC: log fold change

Article Snippet: Up to 5E6 cells were then resuspended in 100 μl Opti-MEM and electroporated with 10 μg CleanCap™ Cas9 mRNA (TriLink, Cat-no. L-7206).

Techniques: Electroporation, Selection, Transduction, In Vivo, Isolation, Produced, In Vitro, Comparison, Gene Knockout

Journal: Circulation Research

Article Title: RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function

doi: 10.1161/CIRCRESAHA.122.321728

Figure Lengend Snippet: Human RBPMS2 - (RNA-binding protein with multiple splicing [variants] 2)-null cardiomyocytes exhibit myofibrillar disarray and calcium handling defects similar to those in zebrafish rbpms2 -null cardiomyocytes. A , Schematic diagrams of human RBPMS2 ( top ) and the predicted protein product of the Δ RBPMS2 null allele created with CRISPR/Cas9-mediated genome editing ( bottom ). The asterisk shows the location of a premature stop codon within the RRM (RNA-recognition motifs) caused by a frame-shifting 2 base pair deletion. The white box shows the location of divergent amino acids before the stop codon. B and C , Bar graphs showing the relative expression levels of RBPMS2 in wild-type control (CTRL) and ΔRBPMS2 human induced pluripotent stem cells (hiPSCs; B ) and cardiomyocytes (hiPSC-CMs; C ) after 15 days of directed differentiation. n=6 biological replicates/group, each with three technical replicates. Error bars show one standard deviation. Statistical significance was determined by an unpaired, 2-tailed Student's t test assuming equal variances. Significant P values are shown. D–K , Representative confocal projections of CTRL ( D–G ) and ΔRBPMS2 ( H–K ) hiPSC-CMs immunostained with antibodies that detect cardiac troponin T (CT3; green) or alpha-actinin (anti-sarcomeric alpha-actinin; magenta) to visualize thin filaments or Z-disks, respectively, and counterstained with DAPI (4'‚6-diamidino-2-phenylindole; blue). Single ( D–F; H–J ) and merged triple ( G and K ) channel images are shown. n=50 cells/group from at least 3 wells from 2 separate differentiations. L and M , Representative traces of fluorescence intensity over baseline (F/F o ; top ) derived from line scans ( bottom ) of CTRL ( L ) and ΔRBPMS2 ( M ) hiPSC-CMs loaded with fluo-4 and paced at 1 Hz. N–S , Violin plots showing calcium transient amplitude ( N ), duration at 80% (CaTD80; O ) and 50% (CaTD50; P ) repolarization, time-to-peak amplitude ( Q ), upstroke velocity ( R ), and unpaced spontaneous beating rate ( S ). n=80 cells/group, 40 each from 2 separate differentiations. Statistical significance was determined by an unpaired, 2-tailed Student's t test assuming equal variances or a Mann-Whitney U test. Significant P values are shown. Scale bars=25 μm.

Article Snippet: We previously described methods to generate a doxycycline-inducible Cas9 line in the WTC-11 iPSC background (Coriell Institute).

Techniques: RNA Binding Assay, CRISPR, Expressing, Control, Standard Deviation, Fluorescence, Derivative Assay, MANN-WHITNEY

Journal: International journal of molecular sciences

Article Title: Knockout of the Cardiac Transcription Factor NKX2-5 Results in Stem Cell-Derived Cardiac Cells with Typical Purkinje Cell-like Signal Transduction and Extracellular Matrix Formation.

doi: 10.3390/ijms241713366

Figure Lengend Snippet: Figure 1. Schematic representation of the CrispR-Cas9 guided knockout of NKX2-5 in hiPSC using the pCAS-Guide vector as performed in this study. The pCAS-Guide vector and the donor DNA Luci- Puro were transfected via lipofection into SFS.1WT hiPSC. The pCAS-Guide controlled expression of Cas9 allows for the targeted knockout of the NKX2-5 gene and the integration of Luci-Puro into the accessible genome of the SFS.1WT cell.

Article Snippet: 2023, 24, 13366 10 of 17 Cas9 NKX2-5 Knockout Kit (Origene (Rockville, MD, USA) #KN511024) as described in the manufacturers’ instructions.

Techniques: CRISPR, Knock-Out, Plasmid Preparation, Transfection, Expressing

Journal: International journal of molecular sciences

Article Title: Knockout of the Cardiac Transcription Factor NKX2-5 Results in Stem Cell-Derived Cardiac Cells with Typical Purkinje Cell-like Signal Transduction and Extracellular Matrix Formation.

doi: 10.3390/ijms241713366

Figure Lengend Snippet: Figure 2. Generation of an NKX2-5KO hiPSC-line. (A) Exemplary hiPSC colony after 17 days of selection with Puromycin. (B) hiPSC culture of isolated, CrispR-Cas9-treated hiPSC colonies. The cells were differentiated towards hiPSC-derived cardiomyocytes. The pictures show the cells as hiPSC, as mesodermal/cardiac cells at day 4 of differentiation and as young cardiomyocytes at day 8 of differentiation. (C) Young cardiomyocytes derived from CrispR-Cas9-treated hiPSC were isolated at day 8 of differentiation and analysed for the expression of NKX2-5 via PCR. Clones 1, 3, 4, 5 show an amplified NKX2-5 fragment. Clone 2 lacks the NKX2-5 fragment at 146 bp (green square).

Article Snippet: 2023, 24, 13366 10 of 17 Cas9 NKX2-5 Knockout Kit (Origene (Rockville, MD, USA) #KN511024) as described in the manufacturers’ instructions.

Techniques: Selection, Isolation, CRISPR, Derivative Assay, Expressing, Clone Assay