Journal: bioRxiv

Article Title: Expanding the toolkit for genetic manipulation and discovery in Candida species using a CRISPR ribonucleoprotein-based approach

doi: 10.1101/2023.06.16.545382

Figure Lengend Snippet: The CRISPR-Cas9 RNP system using 130-150 bp homology regions efficiently generates ADE2 deletions in C. glabrata using NatMX and HphMX . (A) Schematic of pAG25 NatMX plasmid. P1 and P2 indicate location of amplification sequences. (B) Schematic of pAG32 HphMX plasmid. P1 and P2 indicate location of amplification sequences. (C) Representative transformation plate for ADE2 deletion using NatMX with and without addition of CRISPR-RNP. (D) Total number of positive transformants using NatMX with and without addition of CRISPR- RNP. Numbers represent the summation across three separate transformations. (E) Total number of positive transformants using HphMX with and without addition of CRISPR-RNP. Numbers represent the summation across three separate transformations.

Article Snippet: Custom Alt-R CRISPR gRNAs were designed and ordered from Integrated DNA Technologies ( ).

Techniques: CRISPR, Plasmid Preparation, Amplification, Transformation Assay

Journal: bioRxiv

Article Title: Expanding the toolkit for genetic manipulation and discovery in Candida species using a CRISPR ribonucleoprotein-based approach

doi: 10.1101/2023.06.16.545382

Figure Lengend Snippet: The CRISPR-Cas9 RNP system generates single and double gene deletions utilizing NatMX and HphMX in C. glabrata . (A and B) Five-fold serial dilution spot assays with and without 64 μg/mL fluconazole (FLZ). Indicated single deletion strains were generated using the CRISPR-Cas9 RNP method. Double deletion strains were generated using CRISPR-Cas9 RNP method sequentially and three independent clones are shown. Images were captured at 48 hours. (C and D) Expression of the indicated genes were determined by qRT-PCR analysis of mid-log phase cells. Data was normalized to RDN18 mRNA levels and are the average of three biological replicates with three technical replicates each. Error bars represent the standard deviation.

Article Snippet: Custom Alt-R CRISPR gRNAs were designed and ordered from Integrated DNA Technologies ( ).

Techniques: CRISPR, Serial Dilution, Generated, Clone Assay, Expressing, Quantitative RT-PCR, Standard Deviation

Journal: bioRxiv

Article Title: Expanding the toolkit for genetic manipulation and discovery in Candida species using a CRISPR ribonucleoprotein-based approach

doi: 10.1101/2023.06.16.545382

Figure Lengend Snippet: The CRISPR-Cas9 RNP system efficiently generates gene deletions utilizing BleMX in C. glabrata . (A) Schematic of pCY3090-07 plasmid. P1 and P2 indicate location of amplification primer sequences. (B) Total number of positive transformants using BleMX with and without addition of CRISPR-Cas9 RNP. Numbers are the summation across three separate transformations. (C) Five-fold serial dilution spot assays of indicated strains with and without 64 μg/mL fluconazole (FLZ). Two independent clones are shown for erg3Δ ( BleMX ). Images were captured at 48 hours.

Article Snippet: Custom Alt-R CRISPR gRNAs were designed and ordered from Integrated DNA Technologies ( ).

Techniques: CRISPR, Plasmid Preparation, Amplification, Serial Dilution, Clone Assay

Journal: bioRxiv

Article Title: Expanding the toolkit for genetic manipulation and discovery in Candida species using a CRISPR ribonucleoprotein-based approach

doi: 10.1101/2023.06.16.545382

Figure Lengend Snippet: The CRISPR-RNP system efficiently generates gene deletions utilizing KanMX for C. glabrata . (A) Schematic of pUG6 plasmid. P1 and P2 indicate location of amplification primer sequences. (B) Total number of positive transformants using KanMX with and without addition of CRISPR-RNP. Numbers are the summation across three separate transformations. (C) Five- fold serial dilution spot assays of indicated strains with and without 64 μg/mL fluconazole (FLZ). Images were captured at 48 hours.

Article Snippet: Custom Alt-R CRISPR gRNAs were designed and ordered from Integrated DNA Technologies ( ).

Techniques: CRISPR, Plasmid Preparation, Amplification, Serial Dilution

Journal: bioRxiv

Article Title: Expanding the toolkit for genetic manipulation and discovery in Candida species using a CRISPR ribonucleoprotein-based approach

doi: 10.1101/2023.06.16.545382

Figure Lengend Snippet: The CRISPR-Cas9 RNP system generates endogenous epitope tagged proteins using KanMX in C. glabrata . (A) Schematic of pFA6-3HA-KanMX plasmid. P1 and P2 indicate location of amplification primer sequences. (B and C) Indicated strains were either untreated (-) or treated (+) with 64 μg/mL of fluconazole (FLZ) for three hours. Whole cell extracts were isolated and immunoblotted against HA antibody for detection of Erg3 or Erg11. Histone H3 was used as a loading control. Three independent clones were represented for Erg3-3xHA and Erg11-3xHA. (D and E) Five-fold serial dilution spot assays of indicated strains with 0, 16, and 64 μg/mL fluconazole (FLZ), respectively. Three independent clones were represented for Erg3-3xHA and Erg11-3xHA. Images were captured at 48 hours.

Article Snippet: Custom Alt-R CRISPR gRNAs were designed and ordered from Integrated DNA Technologies ( ).

Techniques: CRISPR, Plasmid Preparation, Amplification, Isolation, Control, Clone Assay, Serial Dilution

Journal: bioRxiv

Article Title: Expanding the toolkit for genetic manipulation and discovery in Candida species using a CRISPR ribonucleoprotein-based approach

doi: 10.1101/2023.06.16.545382

Figure Lengend Snippet: The CRISPR-Cas9 RNP system generates gene deletions using a codon optimized BleMX in C. auris . (A) Schematic of pCdOpt-BMX plasmid. P1 and P2 indicate location of amplification primer sequences. (B) Five-fold serial dilution spot assays of indicated C. auris strains with and without 64 μg/mL fluconazole (FLZ). Four independent clones were represented for Caurerg3Δ strain ( BleMX ). Images were captured at 48 hours.

Article Snippet: Custom Alt-R CRISPR gRNAs were designed and ordered from Integrated DNA Technologies ( ).

Techniques: CRISPR, Plasmid Preparation, Amplification, Serial Dilution, Clone Assay

Journal: bioRxiv

Article Title: Expanding the toolkit for genetic manipulation and discovery in Candida species using a CRISPR ribonucleoprotein-based approach

doi: 10.1101/2023.06.16.545382

Figure Lengend Snippet: The CRISPR-Cas9 RNP system is used for deleting SET1 in C. albicans . (A) Schematic of pBSS2- SAT1- FLP plasmid. P1 and P2 indicate location of amplification primer sequences. (B) Whole cell extracts were isolated from indicated C. albicans strain SC5314 and immunoblotted against methyl-specific H3K4 mono-, di- and trimethylation antibodies. Histone H3 was used as a loading control. (C) Five-fold serial dilution spot assays of indicated C. albicans strains with and without 0.5 µg/mL fluconazole (FLZ). Images were captured at 24 hours.

Article Snippet: Custom Alt-R CRISPR gRNAs were designed and ordered from Integrated DNA Technologies ( ).

Techniques: CRISPR, Plasmid Preparation, Amplification, Isolation, Control, Serial Dilution

Journal: Blood

Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

doi: 10.1182/blood.2023021991

Figure Lengend Snippet: lncRNA dropout CRISPR-Cas9 screen in MM cell lines. (A) Schematic representation of the CRISPR screening pipeline. (B) Spearman's correlation between pgRNA read count profiles (from DNA collected 30 days after transduction and selection of the library) across screen replicates = 0.85 and 0.88, respectively, for AMO-1 and ABZB, with color bars on top/left indicating cluster membership obtained via hierarchical clustering (complete distance method). (C) Representation of pgRNA abundance log fold changes (logFCs) in DNA collected 30 days after library transduction and selection vs plasmidic amounts for 3 groups of pgRNAs: nontargeting (negative controls [median logFC = 0.27 and 0.35, respectively, for AMO-1 and ABZB]), targeting ribosomal protein genes (control essential genes, median logFC = −0.68 and −0.43, with a logFC ≤−0.5, corresponding to a MAGeCK FDR ≤20%), and lncRNAs, across the 2 screens. Each point represents 1 of the 12 472 pgRNAs in the library with coordinates on the y-axis indicating the median logFC across screen replicates. (D) Gene-wise MAGeCK robust rank aggregation (RRA) scores for significant dependencies identified in the 2 screens at an FDR ≤20%. Top essential control genes, dependencies that are private to each cell line and shared across them (as per the color scheme) are highligted. (E) Number of significantly essential lncRNAs (at an FDR ≤20%) in the 2 screened cell lines and their overlap. MOI, multiplicity of infection.

Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

Techniques: CRISPR, Transduction, Selection, Control, Infection

Journal: Blood

Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

doi: 10.1182/blood.2023021991

Figure Lengend Snippet: Functional validation of prioritized oncogenic lncRNA candidates. (A) RP11-350G8.5 and LINC00467 basal expression levels via quantitative real time PCR (qRT-PCR) in MM cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors (values are normalized to the expression of GAPDH). (B) Representative image of genomic PCR products before and after KO of LINC00467 and RP11-350G8.5 in AMO-1 cells, visualized on 1.5% agarose gels. On the right: Sanger sequence of the amplicons encompassing the CRISPR-targeted region. Blue rectangles highlight pgRNA binding sites, whereas colored lines refer to the schematic picture of the KO reported above the gel picture (on the left). (C) Representative image of flow cytometric monitoring of AMO-1 and ABZB cells transduced with a SCRAMBLE-GFP-CRISPR vector (dark gray) or LINC00467/KO-GFP-CRISPR vector (light blue) or RP11-350G8.5/KO- GFP-CRISPR vector (red). Light-gray curves represent the percentage of viable cells at day 0 (48 hours after lentiviral transduction) with overlapping colored curves at day 20. (D) Representative images of colony assay of AMO-1 and ABZB GFP-sorted cells, 15 days after plating, were generated using EVOS XL-Core microscope (Invitrogen by Thermo Fisher) (magnification ×10). (E) Number of colonies in 3 independent wells. (F) Dose-response curves 24 hours after treatment with bortezomib (1-10 nM). Percentage of viable cells ± standard deviation are normalized with respect to DMSO-treated cells (vehicle) for each experimental condition. Statistical differences were assessed across all plots via Student t test; ∗ P < .05, ∗∗ P < .01, and ∗∗∗ P < .001.

Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

Techniques: Functional Assay, Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Sequencing, CRISPR, Binding Assay, Transduction, Plasmid Preparation, Colony Assay, Generated, Microscopy, Standard Deviation

Journal: Blood

Article Title: An unbiased lncRNA dropout CRISPR-Cas9 screen reveals RP11-350G8.5 as a novel therapeutic target for multiple myeloma ∗

doi: 10.1182/blood.2023021991

Figure Lengend Snippet: RP11-350G8.5 putative oncogenic role: in vitro validation and preliminary data from in vivo models. (A) Flow cytometric monitoring of GFP expression in ABZB cells transduced with a SCRAMBLE-GFP-CRISPR negative control vector, an RPL8 /KO-GFP-CRISPR positive control vector (selected from Project Score [37]), and 2 GFP-CRISPR constructs encoding for 2 pgRNAs targeting RP11-350G8.5. Gray curves represent the percentage of viable cells at day 0 (48 hours after lentiviral transduction), while colored curves represent the percentage of viable cells at day 20. Bars on the right represent the fold change in percentage of GFP-expressing cells 20 days after target depletion against day 0. (B) Evaluation of IL-6R RNA expression level through quantitative real time PCR (qRT-PCR) on ABZB after transduction with SCRAMBLE vector or KO of RP11-350G8.5 with pgRNA#1 or pgRNA#2 or with a vector overexpressing RP11-350G8.5 (UP). (Data are normalized to the expression of GAPDH.) Statistics were obtained using multiple t -tests, resulting in no significant (ns) differences, as per the reported P values. (C) Flow cytometric monitoring of GFP in JJN.3 and NCI-H929 MM transduced cells, and percentage of GFP-positive cells is reported by overlapping curves referred to day 20 (colored curves) against day 0 (light gray curves). (D) Validation of RP11-350G8.5 KO in nontumoral cells, performed as described for A and C. (E) Representative images of RNA-FISH analysis. Nuclei are counterstained with DAPI (blue signal), whereas C3-fluorescein–conjugated GAPDH (green signal) has been used as cytoplasmic marker. Customly designed Stellaris probes targeting RP11-350G8.5 have been conjugated with 5-carboxytetramethylrhodamine (TAMRA) dye (red signal). Representative pictures acquired with a DMI6000-AF6000 Leica (Wetzlar, Germany) fluorescence microscope at magnification ×63 are reported, followed by specific regions of interest (ROIs), which are represented as enlarged images. (F) Dose-response curves 24 hours after treatment with bortezomib in AMO-1 cells overexpressing RP11-350G8.5 (1-10 nM). Statistics were analyzed using multiple t -tests (cutoff ∗ P < .05, ∗∗ P < .01). (G) In vivo imaging of engrafted ABZB cells. A total of 5 × 10 6 ABZB cells, which previously underwent highly efficient transduction (multiplicity of infection = 1) of RP11-350G8.5 KO-GFP or the SCRAMBLE vectors, were subcutaneously inoculated in mice (n = 2 per group). Images of tumors were acquired when the tumoral masses became palpable (identified as DAY 1), and at the end of the experiment (DAY 16, when tumors reached 2 cm in diameter). Both DAY 1 and DAY 16 were set up by considering SCRAMBLE mice, because SCRAMBLE cells have been faster to generate tumoral masses, due to their higher proliferative rate, and to grow up to 2 cm in diameter, with respect to KO cells. Tumors appear as yellow high-density signals on the right flank of the mice. Pictures were obtained with the IVIS (Perkin Elmer) system. (H) Tumor growth as mean measurement ± standard deviation (SD) across mice groups (n = 2). (I) Photographs of excised tumors were captured by a digital camera. (J) Weights of excised tumors, reported as mean ± SD across mice groups. Statistics were analyzed using multiple t -tests (cutoff: ∗ P < .05).

Article Snippet: The human paired-guide RNA (pgRNA) library pool ( ) (Addgene number 89640) was used to perform the CRISPR-Cas9 screens (supplemental Materials and methods; , and ).

Techniques: In Vitro, Biomarker Discovery, In Vivo, Expressing, Transduction, CRISPR, Negative Control, Plasmid Preparation, Positive Control, Construct, RNA Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Marker, Fluorescence, Microscopy, In Vivo Imaging, Infection, Standard Deviation

Journal: Chinese Medical Journal

Article Title: UBE2C affects breast cancer proliferation through the AKT/mTOR signaling pathway

doi: 10.1097/CM9.0000000000001708

Figure Lengend Snippet: Suppressing UBE2C expression affects BC cell proliferation and invasion. (A) mRNA and protein expression levels of UBE2C detected after siRNA transfection in MDA-MB-231 and MCF-7 cells; (B) CCK-8 assay was performed to assess cell proliferation in MDA-MB-231 and MCF-7 cells transfected with UBE2C siRNA (siUBE2C-1 and siUBE2C-2) and control siRNA. ∗ P < 0.050; (C) Knockdown of UBE2C significantly decreased the invasion of two cell lines (original magnification × 400). † P < 0.010, ‡ P < 0.001. BC: Breast cancer; UBE2C: Ubiquitin-conjugating enzyme E2C.

Article Snippet: When the tumor cells reached the required confluency, we treated them with small interfering RNA (siRNA) (Ribobio, Guangzhou, China) mixed with Lipofectamine 2000 transfection reagent (Invitrogen, CA, USA).

Techniques: Expressing, Transfection, CCK-8 Assay, Control, Knockdown, Ubiquitin Proteomics

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA TOB1-AS1, an epigenetically silenced gene, functioned as a novel tumor suppressor by sponging miR-27b in cervical cancer

doi:

Figure Lengend Snippet: TOB1-AS1 inhibited cell growth and invasion. A. PCR assay was used to verify the expression of TOB1-AS1 in cells. B. Overexpression of TOB1-AS1 in HeLa cells suppressed proliferation, while knockdown of TOB1-AS1 promoted proliferation of C33A cells. C. Upregulation of TOB1-AS1 induced cell cycle arrest at G1 phase; Silencing of TOB1-AS1 promoted cell cycle progression from G1 phase to S phase. D. Apoptotic cells were increased by pcTOB1-AS1 and decreased by si-RNA1 and si-RNA2. E. The invasion ability were assayed using the transwell assay in HeLa and C33A cells. pcDNA3.1, negative control; pcTOB1-AS1, TOB1-AS1 expressing plasmid; si-ctrl, siRNA control; si-RNA1/2, siRNAs for knocking down TOB1-AS1. *P<0.05; **P<0.01.

Article Snippet: Plasmid construction and oligonucleotide transfection Small interfering RNA (siRNA) and non-specific control siRNA (si-ctrl) were synthesized by GenePharma Co., Ltd. (Shanghai, China) and transfected into cells by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).

Techniques: Expressing, Over Expression, Knockdown, Transwell Assay, Negative Control, Plasmid Preparation, Control

Journal: American Journal of Cancer Research

Article Title: Long noncoding RNA TOB1-AS1, an epigenetically silenced gene, functioned as a novel tumor suppressor by sponging miR-27b in cervical cancer

doi:

Figure Lengend Snippet: TOB1-AS1/miR-27b axis was involved in cell growth in vivo. (A) The protein levels of Cyclin D1, Bax, E-cadherin, and N-cadherin were detected by western blot analysis. (B) Representative xenograft tumors obtained from mouse injected with pcTOB1-AS1 or pcDNA3.1. (C and D) Construction of TOB1-AS1 stably expressing cells led to an decreasing of tumor volume (C) and weight (D). (E and F) The expression of TOB1-AS1 (E) and miR-27b (F) in xenograft tumors were measured by qPCR. (G) A schematic diagram deciphering the mechanism underlying the effect of epigenetically silenced TOB1-AS1 on the expression of miR-27b and cervical cancer progression. mim con, mimic control; inh con, inhibitor control. pcTOB1-AS1, TOB1-AS1 expressing plasmid; si-ctrl, siRNA control; si-RNA1, siRNA for knocking down TOB1-AS1; **P<0.01.

Article Snippet: Plasmid construction and oligonucleotide transfection Small interfering RNA (siRNA) and non-specific control siRNA (si-ctrl) were synthesized by GenePharma Co., Ltd. (Shanghai, China) and transfected into cells by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).

Techniques: In Vivo, Western Blot, Injection, Stable Transfection, Expressing, Control, Plasmid Preparation