Journal: Journal of Bacteriology

Article Title: Conditional Mutation of an Essential Putative Glycoprotease Eliminates Autolysis in Staphylococcus aureus

doi: 10.1128/jb.01806-06

Figure Lengend Snippet: FIG. 2. Triton X-100-induced autolysis of conditional gcp mutants. (A) spacp-regulated gcp mutant RN4220/Pspac-gcp; (B) gcp antisense mutant WCUH29/gcp-as. The indicated strains and the control, WCUH29/pYH4, were grown in TSB in the presence of different concentrations of IPTG (1 mM) or ATc (0.5 g/ml). Results were normalized to the OD580 at time zero (OD0). The percent lysis was determined as follows: percent lysis at time t [(OD0 ODt)/OD0] 100. The experiments were repeated at least three times. Each figure represents the results of one experiment.

Article Snippet: The resulting plasmid, pLZ106, was purified and electroporated into S. aureus WCUH29, resulting in strain WCUH29/pLZ106. lux expression was monitored with a Chiron luminometer.

Techniques: Mutagenesis, Control, Lysis

Journal: Journal of Bacteriology

Article Title: Conditional Mutation of an Essential Putative Glycoprotease Eliminates Autolysis in Staphylococcus aureus

doi: 10.1128/jb.01806-06

Figure Lengend Snippet: FIG. 3. Effects of penicillin G on the growth of conditional gcp mutants. (A) spacp-regulated gcp mutant RN4220/Pspac-gcp; (B) gcp antisense mutant WCUH29/gcp-as. The indicated strains and the con- trol, WCUH29/pYH4, were grown in TSB in the presence of different concentrations of inducer (either IPTG or ATc). Penicillin (20 MIC) was added to the exponential-phase cultures at a final concentration of 8 g/ml. The bacterial cultures were continuously incubated, and the OD600 values for the cultures were measured every hour for 8 h. FIG. 4. Effect of down-regulation of gcp expression on hydrolase activity. The spacp-regulated gcp mutant was grown in TSB, with or without the inducer IPTG (1 mM), and the bacterial cells were heat killed, collected by centrifugation, and washed with H2O. (A) The heat-killed cells were resuspended with fresh filter-sterilized superna- tants of overnight cultures of RN4220. The optical density of resus- pended dead cells was adjusted to 0.5 at 600 nm, and the dead cells were incubated at 37°C with shaking. The OD600 was measured every 30 min. (B and C) Zymogram analysis of the spacp-regulated gcp mutant. Equal amounts (10 g) of proteins prepared from the super- natants of different cultures of S. aureus strains were loaded and separated in 10% SDS-PAGE gels containing 0.2% heat-killed cells harvested from culture with 1 mM IPTG (B) or without IPTG (C). Lytic bands appeared as dark zones after scanning. M, protein molec- ular size marker.

Article Snippet: The resulting plasmid, pLZ106, was purified and electroporated into S. aureus WCUH29, resulting in strain WCUH29/pLZ106. lux expression was monitored with a Chiron luminometer.

Techniques: Mutagenesis, Concentration Assay, Incubation, Expressing, Activity Assay, Centrifugation, SDS Page, Marker

Journal: Journal of Bacteriology

Article Title: Conditional Mutation of an Essential Putative Glycoprotease Eliminates Autolysis in Staphylococcus aureus

doi: 10.1128/jb.01806-06

Figure Lengend Snippet: FIG. 6. Expression of lux driven by the cidA promoter in the gcp antisense expression strain WCUH29/gcp-as. Promoter activation was represented as the mean light intensity/OD600 ratio from triplicate readings at different times during growth. RLU, relative light units.

Article Snippet: The resulting plasmid, pLZ106, was purified and electroporated into S. aureus WCUH29, resulting in strain WCUH29/pLZ106. lux expression was monitored with a Chiron luminometer.

Techniques: Expressing, Activation Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CRISPR-Cas9 homology-independent targeted integration of exons 1–19 restores full-length dystrophin in mice

doi: 10.1016/j.omtm.2023.08.009

Figure Lengend Snippet: Correction of a Dmd mutation in neonatal mice through homology-independent targeted integration (A) The gene editing system was delivered via intraperitoneal injection in neonatal mice (n = 3–5 per group) in a 1:1 ratio of the Cas9 and donor vectors at a total dose of 2.9×10 14 vector genomes per kg. Tissues were collected for analysis 4 weeks later. (B) There was a trend toward greater numbers of AAV genomes detected by droplet digital PCR in the heart than in skeletal muscles, but this did not reach statistical significance. (C and D) Gene editing efficiency and transcript correction, measured by droplet digital PCR, were highest in the heart. (E) Representative 0.5-mm regions of interest from the heart stained for dystrophin are shown (top row), along with automated quantification of muscle fiber dystrophin positivity (bottom row). Scale bar represents 100 μm. (F) Dystrophin positivity analysis demonstrated an increase in PDPF in the heart. (G) There was no significant increase in dystrophin expression when measured by capillary western immunoassay. All data is presented as mean ± standard deviation. AAV, adeno-associated virus; Dia, diaphragm; TA, tibialis anterior; vg/dg, vector genomes per diploid genome; PDPF, percent dystrophin-positive fibers; WT, wild-type. ∗p < 0.05 vs. untreated group.

Article Snippet: Sections were incubated for 2 h at room temperature using primary antibodies against laminin (MAB4656 rat monoclonal antibody, R&D Systems, 1:400 dilution) and dystrophin (ab154168 rabbit monoclonal antibody, 1:400 dilution) or Cas9 (#51610 rabbit monoclonal antibody, Cell Signaling Technologies, 1:200 dilution) in blocking buffer.

Techniques: Mutagenesis, Injection, Plasmid Preparation, Digital PCR, Muscles, Staining, Expressing, Western Blot, Standard Deviation, Virus

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CRISPR-Cas9 homology-independent targeted integration of exons 1–19 restores full-length dystrophin in mice

doi: 10.1016/j.omtm.2023.08.009

Figure Lengend Snippet: Evaluation of different Cas9:donor AAV vector ratios for homology-independent targeted integration-based gene editing (A) Neonatal mice (n = 4–5 per group) were injected with different ratios of the Cas9 and donor AAV vectors at a total dose of 2.0×10 14 vector genomes per kg. (B) More AAV genomes were detected in the heart than TA, with highly variable results in the diaphragm. (C) Maximal knockin efficiency was achieved in the heart in the 1:5 group. (D) Maximal transcript correction was likewise achieved in the heart. (E) A significant increase in dystrophin expression was seen in the heart, as measured by capillary western immunoassay. (F) Automated quantitative immunofluorescence analysis showed an increase in PDPF in the heart. (G) There was no significant increase in mean fiber staining intensity. (H) Representative 0.5-mm regions of interest from the heart stained for dystrophin are shown (top row), along with automated quantification of muscle fiber dystrophin positivity (bottom row). Scale bar represents 100 μm. All data is presented as mean ± standard deviation. AAV, adeno-associated virus; Dia, diaphragm; TA, tibialis anterior; vg/dg, vector genomes per diploid genome; PDPF, percent dystrophin-positive fibers; WT, wild-type. ∗p < 0.05 vs. untreated group; #p < 0.05 vs. 5:1 group, †p < 0.01 vs. 1:1 group.

Article Snippet: Sections were incubated for 2 h at room temperature using primary antibodies against laminin (MAB4656 rat monoclonal antibody, R&D Systems, 1:400 dilution) and dystrophin (ab154168 rabbit monoclonal antibody, 1:400 dilution) or Cas9 (#51610 rabbit monoclonal antibody, Cell Signaling Technologies, 1:200 dilution) in blocking buffer.

Techniques: Plasmid Preparation, Injection, Knock-In, Expressing, Western Blot, Immunofluorescence, Staining, Standard Deviation, Virus

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CRISPR-Cas9 homology-independent targeted integration of exons 1–19 restores full-length dystrophin in mice

doi: 10.1016/j.omtm.2023.08.009

Figure Lengend Snippet: Correction of a Dmd mutation in adult mice through homology-independent targeted integration (A) The gene editing system was delivered by tail vein injection in 8-week-old adult mice (n = 3–5 per group) in a 1:5 ratio of the Cas9 to donor vectors at a total dose of 2.0×10 14 vector genomes per kg. (B) There was a trend toward more AAV genomes detected in the heart than in skeletal muscles, but this did not reach statistical significance. (C) The highest knockin efficiency was seen in the heart. (D) Likewise, the highest efficiency of transcript correction was seen in the heart. (E) Treatment significantly increased dystrophin expression in the heart, as measured by capillary western immunoassay. PDPF (F) and mean fiber dystrophin intensity (G) also increased in the heart in treated mice. (H) Representative 0.5-mm regions of interest from the heart stained for dystrophin are shown (top row), along with automated quantification of muscle fiber dystrophin positivity (bottom row). Scale bar represents 100 μm. All data is presented as mean ± standard deviation. AAV, adeno-associated virus; Dia, diaphragm; TA, tibialis anterior; vg/dg, vector genomes per diploid genome; PDPF, percent dystrophin-positive fibers; WT, wild-type. ∗p < 0.05 vs. untreated group.

Article Snippet: Sections were incubated for 2 h at room temperature using primary antibodies against laminin (MAB4656 rat monoclonal antibody, R&D Systems, 1:400 dilution) and dystrophin (ab154168 rabbit monoclonal antibody, 1:400 dilution) or Cas9 (#51610 rabbit monoclonal antibody, Cell Signaling Technologies, 1:200 dilution) in blocking buffer.

Techniques: Mutagenesis, Injection, Plasmid Preparation, Muscles, Knock-In, Expressing, Western Blot, Staining, Standard Deviation, Virus

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CRISPR-Cas9 homology-independent targeted integration of exons 1–19 restores full-length dystrophin in mice

doi: 10.1016/j.omtm.2023.08.009

Figure Lengend Snippet: Comparison of different promoters driving Cas9 expression (A) Biodistribution of Cas9 expression across the four promoters differed between the different types of muscle but not between treatment groups (n = 4 mice per group). (B and C) The CMV, MHCK7, and SPc5-12 promoters achieved higher knockin and transcript correction efficiencies than CK8. (D and E) The SPc5-12 promoter resulted in the greatest increase in dystrophin expression by immunofluorescence in the heart, while CMV was most effective in the TA. (F) By capillary western immunoassay, increased dystrophin expression could only be detected in the heart. (G) Representative regions of interest from the heart (0.5 mm) and TA (1 mm) stained for dystrophin are shown. Scale bar represents 100 μm (heart) or 200 μm (TA). All data is presented as mean ± standard deviation. AAV, adeno-associated virus; Dia, diaphragm; TA, tibialis anterior; vg/dg, vector genomes per diploid genome; PDPF, percent dystrophin-positive fibers; WT, wild-type. ∗p < 0.05 vs. untreated group; #p < 0.05 vs. CK8 group.

Article Snippet: Sections were incubated for 2 h at room temperature using primary antibodies against laminin (MAB4656 rat monoclonal antibody, R&D Systems, 1:400 dilution) and dystrophin (ab154168 rabbit monoclonal antibody, 1:400 dilution) or Cas9 (#51610 rabbit monoclonal antibody, Cell Signaling Technologies, 1:200 dilution) in blocking buffer.

Techniques: Comparison, Expressing, Knock-In, Immunofluorescence, Western Blot, Staining, Standard Deviation, Virus, Plasmid Preparation

Journal: Cancers

Article Title: CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma

doi: 10.3390/cancers11091362

Figure Lengend Snippet: Regorafenib suppresses xenograft tumor growth of the Cas9-expressing hepatocellular carcinoma (HCC) cell line. ( A ) Western blot (WB) analysis of Cas9 protein levels in the HLF cell line. Parent stands for Cas9-nontransduced parental cells, Bulk stands for Cas9-transduced polyclonal cells, and Clone stands for Cas9-transduced monoclonal cells. Relative band intensity is shown. ( B ) Half maximal inhibitory concentration (IC50) values of regorafenib against HLF and HLF-Cas9 cells. ( C ) Nine million Cas9-transduced HLF clone cells per flank were subcutaneously injected into NOD/Shi-scid/IL-2Rγ null (NOG) mice. Once the tumor volume reached 200 mm 3 , mice were treated with either vehicle or 20 mg/kg regorafenib, and tumor volume was measured at various times ( N = 4 for each, * p < 0.05).

Article Snippet: The following primary antibodies were used: Rabbit polyclonal antibody to Bcl-xL (Santa Cruz Biotechnology), rabbit polyclonal antibody to phospho-YAP (Cell Signaling Technology), rabbit polyclonal antibody to YAP (Cell Signaling Technology), rabbit polyclonal antibody to TAZ (Cell Signaling Technology), rabbit polyclonal antibody to ABCB1 (Cell Signaling Technology), rabbit polyclonal antibody to Cas9 (Cell Signaling Technology), rabbit polyclonal antibody to LATS2 (Abcam), and rabbit polyclonal antibody to ACTB (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Western Blot, Concentration Assay, Injection

Journal: Cancers

Article Title: CRISPR Loss-of-Function Screen Identifies the Hippo Signaling Pathway as the Mediator of Regorafenib Efficacy in Hepatocellular Carcinoma

doi: 10.3390/cancers11091362

Figure Lengend Snippet: ( A ) WB analysis of phosphorylated YAP, whole YAP and TAZ protein levels in four human liver cancer cell lines four days after transfection with the NC or LATS2 siRNA. Relative band intensity is shown. ( B , C ) ABCB1 mRNA levels in Hep3B ( B ) and HLF ( C ) cells three days after transfection with the NC or LATS2 siRNA ( N = 4 for each and * p < 0.05). ( D , E ) BCL2L1 mRNA levels in Hep3B ( D ) and HLF ( E ) cells three days after transfection with the NC or LATS2 siRNA ( N = 4 for each and * p < 0.05). ( F ) WB analysis of the protein levels of LATS2, Bcl-xL, and ABCB1 in Hep3B and HLF cells five days after transfection with the NC or LATS2 siRNA. Relative band intensity is shown. ( G ) WB analysis of the protein levels of LATS2, phosphorylated YAP, Bcl-xL and ABCB1 in Cas9-expressing HLF cells transduced with negative control gRNA or LATS2 gRNA. Relative band intensity is shown. ( H ) WB analysis of the protein levels of Bcl-xL and ABCB1 in Hep3B cells treated with either DMSO or 2 μM regorafenib for 48 hours. Relative band intensity is shown.

Article Snippet: The following primary antibodies were used: Rabbit polyclonal antibody to Bcl-xL (Santa Cruz Biotechnology), rabbit polyclonal antibody to phospho-YAP (Cell Signaling Technology), rabbit polyclonal antibody to YAP (Cell Signaling Technology), rabbit polyclonal antibody to TAZ (Cell Signaling Technology), rabbit polyclonal antibody to ABCB1 (Cell Signaling Technology), rabbit polyclonal antibody to Cas9 (Cell Signaling Technology), rabbit polyclonal antibody to LATS2 (Abcam), and rabbit polyclonal antibody to ACTB (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Transfection, Expressing, Transduction, Negative Control