Journal: Cell Host & Microbe

Article Title: Aspergillus fumigatus hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway

doi: 10.1016/j.chom.2023.02.002

Figure Lengend Snippet:

Article Snippet: HCP216549-SG01-3 for generation of p11-KO cell line , GeneCopoiea , Cat# HCP216549-SG01-3.

Techniques: Virus, Recombinant, Cell Culture, Protease Inhibitor, Transfection, SYBR Green Assay, Mass Spectrometry, CyQUANT Assay, LDH Cytotoxicity Assay, cDNA Synthesis, Purification, Gene Expression, Construct, Control, Transformation Assay, Cloning, Plasmid Preparation, Expressing, Software, Imaging, Blocking Assay, Red Blood Cell Lysis, Membrane

Journal: bioRxiv

Article Title: Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope

doi: 10.1101/2023.04.14.536905

Figure Lengend Snippet: a) Nme1Cas9/sgRNA/DNA ternary complex structure, PDB:6JDV. Nme2Cas9 is 98% identical to Nme2Cas9 outside of the WED and PAM-interacting domains. Black spheres represent N- and C-termini and colored spheres represent sites of domain insertion. Deaminase domain insertion sites (Nme2Cas9 aa numbers) are specified to the right, with colors matching the sites indicated in the structure. b) Activities of Nme2-ABE8e constructs in mCherry reporter cells (activated upon A-to-G editing) after plasmid transfection, measured by flow cytometry (n = 3 biological replicates in technical duplicate; data represent mean ± SD). c) A-to-G editing following transfection of Spy-ABE8e vs. Nme2-ABE8e plasmids, using PAM-matched, endogenous HEK293T genomic loci. The editing efficiency at the maximally edited adenine for each target was plotted. Editing efficiencies were measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SD). d) Data from (c) were aggregated and replotted, with each data point representing the maximum A-to-G editing efficiency of an individual target site, as measured by amplicon deep sequencing (n = 3 biological replicates; data represent mean ± SEM). e) Summary of mean A-to-G editing activities and editing windows for Spy- and Nme2-ABE8e constructs in HEK293T cells. Numbers provided for each position in the protospacer represent the mean A-to-G editing efficiency across eight PAM-matched endogenous target sites, as measured via amplicon deep sequencing (n = 3 biological replicates). Crossed-out boxes indicate that no adenine was present at the specified position in the target panel tested.

Article Snippet: U6-driven sgRNA plasmids for the various Cas effectors were cloned using pBluescript sgRNA expression plasmids (Addgene #122089, #122090, #122091 for SpyCas9, SauCas9 and Nme2Cas9 respectively).

Techniques: Construct, Plasmid Preparation, Transfection, Flow Cytometry, Amplification, Sequencing

Journal: Nature Communications

Article Title: Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

doi: 10.1038/s41467-021-22295-w

Figure Lengend Snippet: a Installation (via G•C-to-A•T) of the pathogenic SERPINA1 E342K mutation in HEK293T cells using PE2, PE2*, and Sa KKH PE2*. Editing efficiencies reflect sequencing reads which contain the desired edit. The presence of sgRNAs to promote nicking of the complementary strand is indicated on the x -axis. Results were obtained from three independent experiments and are presented as mean ± SD. b pegRNA used for correction (via A•T-to-G•C) of the E342K mutation includes a spacer sequence, a sgRNA scaffold, an RT template including edited bases (red), and a primer-binding site (PBS). A PAM mutation (AGG to AAG) was introduced to reduce re-cutting of the locus that results in a synonymous codon change. c Evaluating PE expression and subcellular distribution in mouse liver. FVB mice were injected with PE2 or PE2* expression plasmids containing a 3xHA-tag. IHC was performed with an HA-tag antibody. Scale bars: 100 µm (×20 lens). d Average percentage of HA-tag signal from the nucleus. Each dot is the average calculated signal intensity within the nucleus relative to the whole cell from all positive cells in a microscopic image. Numbers are mean ± sem ( n = 20 total images from 3 mice). e Schematic overview of correction strategy of the SERPINA1 E342K mutation in PiZ transgenic mouse model of AATD. Prime editor, pegRNA, and nicking sgRNA plasmid were delivered by hydrodynamic tail-vein injection. f Comparison of the efficiency of K342E correction and indels in mouse livers in PE2 or PE2* treatment groups. Precise editing is defined as the fraction of sequencing reads with both A to G prime editing and synonymous PAM modification. Results were obtained from three mice and presented as mean ± SD. **P < 0.01, * **P < 0.001, * ***P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Backbone plasmids used for pegRNA and sgRNA cloning are available from Addgene: backbone plasmids used for pegRNA and sgRNA cloning are addgene #122089 and 122090.

Techniques: Mutagenesis, Sequencing, Binding Assay, Expressing, Injection, Transgenic Assay, Plasmid Preparation, Comparison, Modification

Journal: Nature Communications

Article Title: Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

doi: 10.1038/s41467-021-22295-w

Figure Lengend Snippet: a pegRNA used for installation (via C•G-to-T•A) of the oncogenic S45F in Ctnnb1 in mouse liver. b Schematic overview of the somatic cell editing strategy to drive tumor formation. Prime editor (PE2 or PE2*), pegRNA for Ctnnb1 S45F, and nicking sgRNA plasmids were delivered by hydrodynamic tail-vein injection along with the MYC transposon and transposase plasmids. c Representative images of tumor burden in mouse liver with PE2 or PE2*. d Tumor numbers in the livers of mice 25 days after injection with PE2 or PE2*. The Control group was pegRNA only. Results were obtained from 4 mice and presented as mean ± SD. e Sanger sequencing from normal liver and representative tumors. The dashed box denotes C to T editing in tumors. *P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. f Schematic of Ctnnb1 S45 deletion strategy using PE2* (S45del). pegRNA used for 3 bp deletion (TCC) is shown. g PE2* treatment leads to oncogenic activation of Ctnnb1 . Prime editor (PE2*), pegRNA ( Ctnnb1 S45del or SERPINA1), and nicking sgRNA plasmids were delivered by hydrodynamic tail-vein injection along with the MYC transposon and transposase plasmids. Mice treated with the peg Ctnnb1 S45del ( n = 4) displayed a large number of liver tumors whereas mice treated with pegSERPINA1 as a control displayed no noticeable oncogenic lesions. beta-Catenin (CTNNB1) IHC staining was performed. Scale bars: 100 µm (×20 lens). h Prime editing efficiency and indels determined by targeted deep sequencing in control liver and representative tumors. Results were obtained from 3 tumors in each group and presented as mean ± SD.

Article Snippet: Backbone plasmids used for pegRNA and sgRNA cloning are available from Addgene: backbone plasmids used for pegRNA and sgRNA cloning are addgene #122089 and 122090.

Techniques: Injection, Control, Sequencing, Activation Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice

doi: 10.1038/s41467-021-22295-w

Figure Lengend Snippet: a Schematic of split-intein dual AAV prime editor. Full-length primer editor (original PE2 ) was reconstituted from two PE2 fragments employing the Npu DNAE split intein . C, carboxy-terminal; N, amino-terminal. b Schematic of the in vivo experiments. Dual AAV8 split-intein prime editor (2 × 10 11 vg total) was delivered to six-week-old PiZ mice by tail-vein injection. Livers were harvested at 2 ( n = 2), 4 ( n = 3), and 10 ( n = 3) weeks after injection and the genomic DNA was isolated for sequencing. c Prime editing efficiency of K342E correction and indels determined by targeted deep sequencing in mouse livers of dual AAV-treated mice. Precise editing is defined as the fraction of sequencing reads with both A to G prime editing and synonymous PAM modification. Results were obtained from two (2 weeks) or three mice (6 and 10 weeks) and presented as mean ± SD. **P < 0.01, * **P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. d Composition of edited alleles at SERPINA1 by UDiTaS analysis. The circle plot shows the fraction of edits that are precise (intended base conversion), small indels (<50 bp) or substitution, deletions between pegRNA and nicking sgRNA sites (<100 bp), large deletions (>100 bp), and AAV fragment insertion. Numbers are average of 3 mice in 10 weeks treated cohort. e The statistically significant large deletion sequences detected by UDiTaS in the 10 weeks treated cohort are displayed as bars spanning the sequence that is deleted (a representative liver of n = 3 mice). Positions of the pegRNA and nicking sgRNA are indicated by dotted lines and the approximate positions of the locus-specific UDiTaS primers are indicated by arrows below the bar chart. The deletion size and number of UMIs associated with each deletion are indicated to the right of each bar. Statistical significance was calculated as a Benjamini–Hochberg adjusted p -value with a cut-off of 0.05.

Article Snippet: Backbone plasmids used for pegRNA and sgRNA cloning are available from Addgene: backbone plasmids used for pegRNA and sgRNA cloning are addgene #122089 and 122090.

Techniques: In Vivo, Injection, Isolation, Sequencing, Modification

Journal: Molecular cell

Article Title: CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons

doi: 10.1016/j.molcel.2017.08.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: B270 (containing sgRNA targeting ATP1A1 + empty sgRNA-expressing cassette) , This paper , Available in Addgene.

Techniques: Virus, Subcloning, Recombinant, Transfection, DNA Extraction, PCR Cloning, Cloning, Plasmid Preparation, Software

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Development of galectin-3 knockdown ARPE-19 cells. ( A , B ) Merged phase-contrast and fluorescent imaging for GFP expression of ARPE-19 cells without ( A ) and with pCRISPR-LGALS3 transfection ( B ). ( C , D ) Immunostaining for galectin-3 of ARPE-19 ( C ) and ARPE-19/LGLAS3 +/− cells ( D ) after FACS and single-cell cultivation. ( E , F ) Phase-contrast image of ARPE-19 ( E ) and ARPE-19/LGLAS3 +/− cells ( F ). Magnification bar in ( A , B ) 100 µm; in ( C , D ) 20 µm, in ( E , F ) 100 µm; blue, Hoechst staining. ( G ) Western blot analysis for galectin-3 of ARPE-19 and ARPE-19/LGLAS3 +/− cells. ( H , I ) Galectin-3 concentration of conditioned cell culture medium ( H ) and cell lysate ( I ) from ARPE-19 and ARPE-19/LGLAS3 +/− cells after incubation in unsupplemented cell culture medium for 72 h. Mean ± SD; ** p < 0.01; **** p < 0.0001; n = 10.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Knockdown, Imaging, Expressing, Transfection, Immunostaining, Staining, Western Blot, Concentration Assay, Cell Culture, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Reduced expression of galectin-3 decreases viability of immortalized RPE cells in vitro. WST-1 assay of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation in cell culture medium without supplementation and various concentrations of hr-galectin-3 (hr-Gal3) for 72 h. Mean ± SD; **** p < 0.0001; n ≥ 28 of at least 4 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, In Vitro, WST-1 Assay, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Decreased expression of galectin-3 declines proliferation of immortalized RPE cells in vitro. BrdU ELISA of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation in cell culture medium without supplementation for 72 h with and without various concentrations of hr-galectin-3 ( A ) and/or an additional treatment with 100mM lactose ( B ). Mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n ≥ 20 of at least 4 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Decreased galectin-3 expression reduces migration of ARPE-19 cells in vitro. Quantification ( A ) and representative images ( B ) of scratch migration assay of native ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation with and without hr-galectin-3 in cell culture medium without supplements for 24 h. ( A ) For quantification, the recolonized area after 24 h was calculated and plotted as the relative recolonized area. ( B ) Representative images of native ARPE-19 (left panel) and ARPE-19/LGALS3 +/− cells (right panel) immediately after scratching (0 h, upper row) and after incubation for 24 h (lower panel). Mean ± SD; *** p < 0.001; **** p < 0.0001; n ≥ 10 of 3 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Migration, In Vitro, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Reduced expression of endogenous galectin-3 enhances cell attachment of immortalized RPE cells in vitro. ( A ) Cell adhesion of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells was quantified 30, 60, 90, 120, 150, and 180 min after seeding and plotted as the relative number of adherent cells. Mean ± SD; n = 12 for each group of 3 independent experiments; * comparison of ARPE-19 versus ARPE-19/LGALS3 +/− cells; # comparison of ARPE-19/FACS versus ARPE-19/LGALS3 +/− cells; **** p < 0.001; #### p < 0.001. ( B ) Relative number of adherent ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells 90 min after seeding and incubation with various concentrations of hr-galectin-3, which was added immediately before seeding. Mean ± SD; n = 15 for each group of 3 independent experiments; ** p < 0.01; **** p < 0.0001.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Cell Attachment Assay, In Vitro, Comparison, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Lack of endogenous galectin-3 promotes epithelial-to-mesenchymal transition of immortalized RPE. ( A – F ) Phaloidin staining (( A , B ) orange) as well as immunofluorescent staining for sm-α-actin (( C , D ) orange) and N-cadherin ( E , F ) green) of ARPE-19 ( A , C , E ) and ARPE-19/LGALS3 +/− cells ( B , D , F ). Scale bar, 20 µm; blue, DAPI staining. ( G ) Real-time rt-PCR for sm-α-actin, E-cadherin, and N-cadherin mRNA expression of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells. Mean ± SD; n ≥ 7 of 5 independent experiments; * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Staining, Quantitative RT-PCR, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Endogenous galectin-3 expression maintains basal pAKT, pERK, and β-catenin signaling in immortalized RPE cells. Western blot analysis ( A ) and densitometry for pAKT ( B ), pERK ( C ), and β-catenin ( D ) expression of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells. Mean ± SD; n ≥ 6 of 6 independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Western Blot