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: Frontiers in Oncology

Article Title: EI24, as a Component of Autophagy, Is Involved in Pancreatic Cell Proliferation

doi: 10.3389/fonc.2019.00652

Figure Lengend Snippet: Loss of EI24 expression in pancreatic cancer cells impairs autophagy. MIA PaCa-2 and Panc-1 cells were transfected with 10 nM control siRNA (siCtrl) or siRNA targeting EI24 or ATG5 for 48 h. (A) The mRNA levels of EI24, ATG5, and GAPDH were analyzed by reverse-transcription PCR. Values represent the expression of EI24 or ATG5 relative to that of GAPDH (with siCtrl values set to 1). (B) The protein levels of EI24 ATG5 and β-actin were analyzed by western blotting. Representative data are shown. Values represent the ratio of the EI24 or ATG5 densitometry value to that of β-actin (with siCtrl values set to 1). (C) EI24 protein expression was observed by immunofluorescence staining using an anti-EI24 antibody. (D) LC3 puncta (autophagosome) were visualized by immunofluorescence staining using an anti-LC3 antibody. (E) Conversion of LC3-I to LC3-II and p62 accumulation were analyzed by western blotting using anti-LC3 and anti-p62 antibodies, respectively. Graphs represent the LC3-II or p62 densitometry value to that of β-actin (with siCtrl values set to 1) from five independent experiments. (F) Co-localization of the autophagosome and lysosome in EI24-knockdown MIA PaCa-2 and Panc-1 cells was analyzed by confocal microscopy using the ZEN 2012 program. Immunofluorescence staining with anti-LC3 (to detect autophagosomes; green dye) and anti-LAMP1 (to detect lysosomes; red dye) antibodies. Co-localization of LC3 with LAMP1 was analyzed by ZEN 2.6 (blue edition). The graph shows the Pearson's co-localization coefficient. (G) MIA PaCa-2 and Panc-1 cells were treated with 100 nM bafilomycin for 30 min, and their protein extracts were analyzed by western blotting using anti-LC3 and β-actin antibodies. Graphs represent the LC3-II densitometry value to that of β-actin (with siCtrl values set to 1) from three independent experiments. Data in (E–G) graphs represent the mean ± standard error of the means (SEM). Comparison were made using Student's t -test, * P < 0.05; ** P < 0.01; n.s., not significant.

Article Snippet: CRISPR EI24 guide RNAs (gRNAs) 1 and 2 in the pLentiCRISPR v2 vector were purchased from GenScript (Piscataway, NJ).

Techniques: Expressing, Transfection, Control, Reverse Transcription, Western Blot, Immunofluorescence, Staining, Knockdown, Confocal Microscopy, Comparison

Journal: Frontiers in Oncology

Article Title: EI24, as a Component of Autophagy, Is Involved in Pancreatic Cell Proliferation

doi: 10.3389/fonc.2019.00652

Figure Lengend Snippet: Loss of EI24 expression in pancreatic cancer cells inhibits cell proliferation. MIA PaCa-2 and Panc-1 cells were transfected with 10 nM control siRNA (siCtrl) and siRNA targeting EI24 or ATG5. (A) After 24 h of transfection, cells were reseeded into a 6-well plate and incubated for 10 days. Then, the cells were fixed and colonies counted. The values represent means ± SEM (Student's t -test, * P < 0.05). (B) After 24 h of transfection, cells were seeded into a 6-well plate and incubated for another 7 days. Cells were fixed and stained with crystal violet. (C) After 24 h of transfection, cells were seeded into a 96-well plate. Images acquired from the IncuCyte instrument at the indicated times were analyzed using the ZOOM 2016 program. Cell confluency was measured in triplicate wells for each sample. The plotted values represent means ± SEM (Student's t -test, *** P < 0.001). (D) Cells transfected with siRNAs were incubated for 48 h. After incubation, the cells were analyzed by flow cytometry to evaluate DNA content. Representative DNA content profiles from three independent experiments are shown. (E) DNA fragmentation in siRNA-transfected MIA PaCa-2 and Panc-1 cell lysates was measured using the Cell Death Detection kit. The y -axes in the graphs indicate the extent of DNA fragmentation. Values plotted in the graphs represent means ± SEM. (Student's t -test, ** P < 0.01; n.s., not significant). (F) Proteins extracted from siRNA-transfected cells was analyzed by western blotting using anti-PARP1 and anti-β-actin antibodies. The proform of PARP (116 kDa, black arrow head) and cleaved PARP (85 kDa, red arrow head) are indicated.

Article Snippet: CRISPR EI24 guide RNAs (gRNAs) 1 and 2 in the pLentiCRISPR v2 vector were purchased from GenScript (Piscataway, NJ).

Techniques: Expressing, Transfection, Control, Incubation, Staining, Flow Cytometry, Western Blot

Journal: Frontiers in Oncology

Article Title: EI24, as a Component of Autophagy, Is Involved in Pancreatic Cell Proliferation

doi: 10.3389/fonc.2019.00652

Figure Lengend Snippet: Loss of EI24 expression in HCT116 cells impairs autophagy but not cell proliferation. HCT116 cells were transfected with 10 nM control siRNA (siCtrl) or siRNA targeting EI24 or ATG5. (A) The mRNA levels of EI24, ATG5, and GAPDH were analyzed by reverse-transcription PCR. (B) The protein levels of EI24 ATG5 and β-actin were analyzed by western blotting. Representative data are shown. Values represent the ratio of the EI24 or ATG5 densitometry value to that of β-actin (with siCtrl values set to 1). Conversion of LC3-I to LC3-II and p62 accumulation were analyzed by western blotting using anti-LC3 and anti-p62 antibodies, respectively. Graphs represent mean ± SEM of the LC3-II and p62 densitometry value to that of β-actin (with siCtrl values set to 1) from three independent experiments. Comparison were made using Student's t -test, * P < 0.05; ** P < 0.01. (C) After 24 h of transfection, cells were seeded into a 6-well plate and incubated for another 7 days. Cells were fixed and stained with crystal violet. (D) After 24 h of transfection, cells were seeded into a 96-well plate. Images acquired from the IncuCyte instrument at the indicated times were analyzed using the ZOOM 2016 program. Cell confluency was measured in triplicate wells for each sample. The plotted values represent means ± SEM. (E) Protein extracts from siRNA-transfected cells were analyzed for DNA fragmentation. The y -axes of the graphs indicate the extent of DNA fragmentation. Values plotted in the graphs represent means ± SEM (Student's t -test, n.s., not significant).

Article Snippet: CRISPR EI24 guide RNAs (gRNAs) 1 and 2 in the pLentiCRISPR v2 vector were purchased from GenScript (Piscataway, NJ).

Techniques: Expressing, Transfection, Control, Reverse Transcription, Western Blot, Comparison, Incubation, Staining

Journal: Frontiers in Oncology

Article Title: EI24, as a Component of Autophagy, Is Involved in Pancreatic Cell Proliferation

doi: 10.3389/fonc.2019.00652

Figure Lengend Snippet: Loss of EI24 expression using CRISPR-Cas9 in MIA PaCa-2 cells decreased cell proliferation. MIA PaCa-2 cells were transfected with CRISPR-Cas9 control (gRNA) and EI24 gRNA (gEI24) using a lentiviral system. (A) After 48 h of incubation, EI24 protein expression was observed by immunofluorescence staining using an anti-EI24 antibody. (B) After 48 h of incubation, the EI24 protein level was observed by western blotting using an anti-EI24 antibody. Conversion of LC3-I to LC3-II and p62 accumulation were analyzed by western blotting using anti-LC3 and anti-p62 antibodies, respectively. Graphs represent the mean ± SEM of EI24, LC3-II, and p62 densitometry value to that of β-actin (with gRNA values set to 1) from three independent experiments. Comparison were made using Student's t -test, * P < 0.05; ** P < 0.01; *** P < 0.001. (C) After 24 h of incubation, cells were seeded into a 96-well plate (1,000 cells/well). Images acquired by the IncuCyte instrument at the indicated times were analyzed using the ZOOM 2016 program. Confluency was measured in triplicate wells for each sample. Values represent the means ± SEM (Student's t -test, * P < 0.05; ** P < 0.01; *** P < 0.001). (D) Control and EI24 gRNA-transfected cells (5 × 10 6 ) were injected into both flanks of Balb/c nude mice. Tumor volume was measured on the indicated days. The y -axes of these graphs represent the fold change in tumor size relative to the initial tumor size. Values represent means ± SEM. (Student's t -test, n.s., not significant, control gRNA mice, n = 5; EI24 gRNA mice, n = 4).

Article Snippet: CRISPR EI24 guide RNAs (gRNAs) 1 and 2 in the pLentiCRISPR v2 vector were purchased from GenScript (Piscataway, NJ).

Techniques: Expressing, CRISPR, Transfection, Control, Incubation, Immunofluorescence, Staining, Western Blot, Comparison, Injection