guide-rna scaffold sequence addgene Search Results


transfection previously characterized gsk3b grna oligonucleotide  (Addgene inc)
91
Addgene inc transfection previously characterized gsk3b grna oligonucleotide
A Fxr1 targeting gRNA sequences and corresponding protospacer adjacent motifs (PAMs). B Evaluation of Fxr1 targeting sgRNAs by SURVEYOR assay 2 days after transfection of sgRNAs and SpCas9 (asterisks indicate the presence of digested bands). C Western blot analysis and quantification of Gsk3β and Fxr1 expression in Neuro2A cells 7 days after transfection of CRISPR/Cas9 constructs (Ctrl n = 6, Fxr1KO n = 7, Student's t ‐test, *** P < 0.001). Bars and error bars are mean ± SEM. D Schematic representation of low‐efficiency transfection of primary neuronal cultures with various plasmids. E–G Evaluation of CRISPR/Cas9 KO of (E) <t>Gsk3b</t> , (F) Fxr1 , and (G) Gsk3b/Fxr1 in primary neuronal cultures by immunostaining. Arrows indicate presence and arrowheads absence of staining. H Quantification of CRISPR/Cas9 KO of Gsk3b and Fxr1 .
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cloning grna targeting exon 1  (Addgene inc)
93
Addgene inc cloning grna targeting exon 1
A Fxr1 targeting gRNA sequences and corresponding protospacer adjacent motifs (PAMs). B Evaluation of Fxr1 targeting sgRNAs by SURVEYOR assay 2 days after transfection of sgRNAs and SpCas9 (asterisks indicate the presence of digested bands). C Western blot analysis and quantification of Gsk3β and Fxr1 expression in Neuro2A cells 7 days after transfection of CRISPR/Cas9 constructs (Ctrl n = 6, Fxr1KO n = 7, Student's t ‐test, *** P < 0.001). Bars and error bars are mean ± SEM. D Schematic representation of low‐efficiency transfection of primary neuronal cultures with various plasmids. E–G Evaluation of CRISPR/Cas9 KO of (E) <t>Gsk3b</t> , (F) Fxr1 , and (G) Gsk3b/Fxr1 in primary neuronal cultures by immunostaining. Arrows indicate presence and arrowheads absence of staining. H Quantification of CRISPR/Cas9 KO of Gsk3b and Fxr1 .
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guide rna  (Addgene inc)
93
Addgene inc guide rna
A Fxr1 targeting gRNA sequences and corresponding protospacer adjacent motifs (PAMs). B Evaluation of Fxr1 targeting sgRNAs by SURVEYOR assay 2 days after transfection of sgRNAs and SpCas9 (asterisks indicate the presence of digested bands). C Western blot analysis and quantification of Gsk3β and Fxr1 expression in Neuro2A cells 7 days after transfection of CRISPR/Cas9 constructs (Ctrl n = 6, Fxr1KO n = 7, Student's t ‐test, *** P < 0.001). Bars and error bars are mean ± SEM. D Schematic representation of low‐efficiency transfection of primary neuronal cultures with various plasmids. E–G Evaluation of CRISPR/Cas9 KO of (E) <t>Gsk3b</t> , (F) Fxr1 , and (G) Gsk3b/Fxr1 in primary neuronal cultures by immunostaining. Arrows indicate presence and arrowheads absence of staining. H Quantification of CRISPR/Cas9 KO of Gsk3b and Fxr1 .
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crispri grnas  (Addgene inc)
94
Addgene inc crispri grnas
A Fxr1 targeting gRNA sequences and corresponding protospacer adjacent motifs (PAMs). B Evaluation of Fxr1 targeting sgRNAs by SURVEYOR assay 2 days after transfection of sgRNAs and SpCas9 (asterisks indicate the presence of digested bands). C Western blot analysis and quantification of Gsk3β and Fxr1 expression in Neuro2A cells 7 days after transfection of CRISPR/Cas9 constructs (Ctrl n = 6, Fxr1KO n = 7, Student's t ‐test, *** P < 0.001). Bars and error bars are mean ± SEM. D Schematic representation of low‐efficiency transfection of primary neuronal cultures with various plasmids. E–G Evaluation of CRISPR/Cas9 KO of (E) <t>Gsk3b</t> , (F) Fxr1 , and (G) Gsk3b/Fxr1 in primary neuronal cultures by immunostaining. Arrows indicate presence and arrowheads absence of staining. H Quantification of CRISPR/Cas9 KO of Gsk3b and Fxr1 .
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lenticas9 grna vectors  (Addgene inc)
93
Addgene inc lenticas9 grna vectors
A Fxr1 targeting gRNA sequences and corresponding protospacer adjacent motifs (PAMs). B Evaluation of Fxr1 targeting sgRNAs by SURVEYOR assay 2 days after transfection of sgRNAs and SpCas9 (asterisks indicate the presence of digested bands). C Western blot analysis and quantification of Gsk3β and Fxr1 expression in Neuro2A cells 7 days after transfection of CRISPR/Cas9 constructs (Ctrl n = 6, Fxr1KO n = 7, Student's t ‐test, *** P < 0.001). Bars and error bars are mean ± SEM. D Schematic representation of low‐efficiency transfection of primary neuronal cultures with various plasmids. E–G Evaluation of CRISPR/Cas9 KO of (E) <t>Gsk3b</t> , (F) Fxr1 , and (G) Gsk3b/Fxr1 in primary neuronal cultures by immunostaining. Arrows indicate presence and arrowheads absence of staining. H Quantification of CRISPR/Cas9 KO of Gsk3b and Fxr1 .
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guide rnas sgrna  (Addgene inc)
96
Addgene inc guide rnas sgrna
A Fxr1 targeting gRNA sequences and corresponding protospacer adjacent motifs (PAMs). B Evaluation of Fxr1 targeting sgRNAs by SURVEYOR assay 2 days after transfection of sgRNAs and SpCas9 (asterisks indicate the presence of digested bands). C Western blot analysis and quantification of Gsk3β and Fxr1 expression in Neuro2A cells 7 days after transfection of CRISPR/Cas9 constructs (Ctrl n = 6, Fxr1KO n = 7, Student's t ‐test, *** P < 0.001). Bars and error bars are mean ± SEM. D Schematic representation of low‐efficiency transfection of primary neuronal cultures with various plasmids. E–G Evaluation of CRISPR/Cas9 KO of (E) <t>Gsk3b</t> , (F) Fxr1 , and (G) Gsk3b/Fxr1 in primary neuronal cultures by immunostaining. Arrows indicate presence and arrowheads absence of staining. H Quantification of CRISPR/Cas9 KO of Gsk3b and Fxr1 .
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grna sequences apc  (Addgene inc)
96
Addgene inc grna sequences apc
A Fxr1 targeting gRNA sequences and corresponding protospacer adjacent motifs (PAMs). B Evaluation of Fxr1 targeting sgRNAs by SURVEYOR assay 2 days after transfection of sgRNAs and SpCas9 (asterisks indicate the presence of digested bands). C Western blot analysis and quantification of Gsk3β and Fxr1 expression in Neuro2A cells 7 days after transfection of CRISPR/Cas9 constructs (Ctrl n = 6, Fxr1KO n = 7, Student's t ‐test, *** P < 0.001). Bars and error bars are mean ± SEM. D Schematic representation of low‐efficiency transfection of primary neuronal cultures with various plasmids. E–G Evaluation of CRISPR/Cas9 KO of (E) <t>Gsk3b</t> , (F) Fxr1 , and (G) Gsk3b/Fxr1 in primary neuronal cultures by immunostaining. Arrows indicate presence and arrowheads absence of staining. H Quantification of CRISPR/Cas9 KO of Gsk3b and Fxr1 .
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grna sublibrary  (Addgene inc)
94
Addgene inc grna sublibrary
Figure 7. The UFMylation pathway contributes to Toxoplasma virulence (A) Rank-ordered plots for in vivo fitness scores from the endomembrane-nucleus <t>sublibrary</t> (left) and the metabolism sublibrary (right). UFMylation-related genes are marked in red. Error bars (gray) represent SEM. (B) Fitness scores of the UFMylation-related genes in Vero cells (in vitro), WT mice (WT), and Ifngr1/ mice (KO) are shown as box plots. Each dot represents fitness scores for individual gRNAs. ***p < 0.001; *p < 0.05 (Wilcoxon rank-sum test). (C) The conserved C-terminal amino acid sequences of the UFM1 homologs from indicated apicomplexan (blue) and model organisms (black). The conserved glycine residue is shown by the arrow. (D) Survival curves of WT mice with intra-footpad infection of 103 tachyzoites of WT (n = 10), DUFM1 (n = 6), DUFM1 + UFM1 (WT) (n = 7), and DUFM1 + UFM1 (G87A) (n = 7). ***p < 0.001; N.S., not significant (log-rank test). (E) Complementations of FLAG-tagged WT and G87A UFM1 protein in DUFM1 parasites. (F) Survival curves of WT (n = 10) or Ifngr1/ (n = 9) mice with intra-footpad infection of 103 tachyzoites of DUFM1 parasites. ***p < 0.001 (log-rank test). See also Figure S6.
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single guide rna sgrna  (Addgene inc)
97
Addgene inc single guide rna sgrna
Figure 7. The UFMylation pathway contributes to Toxoplasma virulence (A) Rank-ordered plots for in vivo fitness scores from the endomembrane-nucleus <t>sublibrary</t> (left) and the metabolism sublibrary (right). UFMylation-related genes are marked in red. Error bars (gray) represent SEM. (B) Fitness scores of the UFMylation-related genes in Vero cells (in vitro), WT mice (WT), and Ifngr1/ mice (KO) are shown as box plots. Each dot represents fitness scores for individual gRNAs. ***p < 0.001; *p < 0.05 (Wilcoxon rank-sum test). (C) The conserved C-terminal amino acid sequences of the UFM1 homologs from indicated apicomplexan (blue) and model organisms (black). The conserved glycine residue is shown by the arrow. (D) Survival curves of WT mice with intra-footpad infection of 103 tachyzoites of WT (n = 10), DUFM1 (n = 6), DUFM1 + UFM1 (WT) (n = 7), and DUFM1 + UFM1 (G87A) (n = 7). ***p < 0.001; N.S., not significant (log-rank test). (E) Complementations of FLAG-tagged WT and G87A UFM1 protein in DUFM1 parasites. (F) Survival curves of WT (n = 10) or Ifngr1/ (n = 9) mice with intra-footpad infection of 103 tachyzoites of DUFM1 parasites. ***p < 0.001 (log-rank test). See also Figure S6.
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human trim28 grna targeting exon 3  (Addgene inc)
91
Addgene inc human trim28 grna targeting exon 3
Figure 7. The UFMylation pathway contributes to Toxoplasma virulence (A) Rank-ordered plots for in vivo fitness scores from the endomembrane-nucleus <t>sublibrary</t> (left) and the metabolism sublibrary (right). UFMylation-related genes are marked in red. Error bars (gray) represent SEM. (B) Fitness scores of the UFMylation-related genes in Vero cells (in vitro), WT mice (WT), and Ifngr1/ mice (KO) are shown as box plots. Each dot represents fitness scores for individual gRNAs. ***p < 0.001; *p < 0.05 (Wilcoxon rank-sum test). (C) The conserved C-terminal amino acid sequences of the UFM1 homologs from indicated apicomplexan (blue) and model organisms (black). The conserved glycine residue is shown by the arrow. (D) Survival curves of WT mice with intra-footpad infection of 103 tachyzoites of WT (n = 10), DUFM1 (n = 6), DUFM1 + UFM1 (WT) (n = 7), and DUFM1 + UFM1 (G87A) (n = 7). ***p < 0.001; N.S., not significant (log-rank test). (E) Complementations of FLAG-tagged WT and G87A UFM1 protein in DUFM1 parasites. (F) Survival curves of WT (n = 10) or Ifngr1/ (n = 9) mice with intra-footpad infection of 103 tachyzoites of DUFM1 parasites. ***p < 0.001 (log-rank test). See also Figure S6.
Human Trim28 Grna Targeting Exon 3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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grna cas9  (Addgene inc)
94
Addgene inc grna cas9
( A ) Workflow of the CRISPR screen in retinal pigment epithelium (RPE1) cells, which were transduced with a lentiviral Genome-Scale CRISPR Knock-out (GeCKO) single <t>guide</t> <t>RNA</t> (sgRNA) library and selected for the sgRNA expression and then survival after treatments with the proteasome inhibitor MG132. Individual surviving cell colonies were collected for sequencing and subsequent analysis. ( B ) Left: The cytotoxicity analysis of wild-type (WT) and DBT knockout (KO) RPE1 cells treated with MG132 at different doses for 96 hr (n=3). Right: The time course analysis of MG132-induced cytotoxicity in the WT and DBT KO cells (n=3). ( C ) Immunoblot analysis of WT RPE1, DBT KO, and DBT’ cells. The DBT’ cells expressed an engineered DBT cDNA that resisted DBT-targeted <t>Cas9</t> cleavage and rescued the DBT expression in the KO cells. ( D ) Cell viability was measured by Calcein-AM staining in WT RPE1, DBT KO, and DBT’ cells treated with MG132 (2 μM, 96 hr). Scale bar, 100 μm. ( E ) Quantification of the cell viability measured by Calcein-AM staining in ( D ) (n=9). ( F ) Left: Immunoblot analysis of RPE1 cells transfected with DBT shRNAs and non-targeting control shRNAs. Right: Quantification of the cell viability under treatment with MG132 (2 μM, 48 hr), as measured by Calcein-AM staining (n=4). ( G ) Immunoblotting and quantification of cleaved PARP as an MG132-induced cell death marker (n=4). ( H ) Immunoblotting and quantification of cleaved Caspase 3 as an MG132-induced cell death marker (n=3). Error bars represent means ± SEM. *p≤0.05; **p≤0.01; ****p≤0.0001. Figure 1—source data 1. Original and uncropped blots for . Figure 1—source data 2. Original and uncropped blots for . Figure 1—source data 3. Original and uncropped blots for . Figure 1—source data 4. Original and uncropped blots for .
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Image Search Results


A Fxr1 targeting gRNA sequences and corresponding protospacer adjacent motifs (PAMs). B Evaluation of Fxr1 targeting sgRNAs by SURVEYOR assay 2 days after transfection of sgRNAs and SpCas9 (asterisks indicate the presence of digested bands). C Western blot analysis and quantification of Gsk3β and Fxr1 expression in Neuro2A cells 7 days after transfection of CRISPR/Cas9 constructs (Ctrl n = 6, Fxr1KO n = 7, Student's t ‐test, *** P < 0.001). Bars and error bars are mean ± SEM. D Schematic representation of low‐efficiency transfection of primary neuronal cultures with various plasmids. E–G Evaluation of CRISPR/Cas9 KO of (E) Gsk3b , (F) Fxr1 , and (G) Gsk3b/Fxr1 in primary neuronal cultures by immunostaining. Arrows indicate presence and arrowheads absence of staining. H Quantification of CRISPR/Cas9 KO of Gsk3b and Fxr1 .

Journal: The EMBO Journal

Article Title: Fxr1 regulates sleep and synaptic homeostasis

doi: 10.15252/embj.2019103864

Figure Lengend Snippet: A Fxr1 targeting gRNA sequences and corresponding protospacer adjacent motifs (PAMs). B Evaluation of Fxr1 targeting sgRNAs by SURVEYOR assay 2 days after transfection of sgRNAs and SpCas9 (asterisks indicate the presence of digested bands). C Western blot analysis and quantification of Gsk3β and Fxr1 expression in Neuro2A cells 7 days after transfection of CRISPR/Cas9 constructs (Ctrl n = 6, Fxr1KO n = 7, Student's t ‐test, *** P < 0.001). Bars and error bars are mean ± SEM. D Schematic representation of low‐efficiency transfection of primary neuronal cultures with various plasmids. E–G Evaluation of CRISPR/Cas9 KO of (E) Gsk3b , (F) Fxr1 , and (G) Gsk3b/Fxr1 in primary neuronal cultures by immunostaining. Arrows indicate presence and arrowheads absence of staining. H Quantification of CRISPR/Cas9 KO of Gsk3b and Fxr1 .

Article Snippet: For primary neuronal culture, transfection previously characterized Gsk3b gRNA oligonucleotide (Khlghatyan et al , ) was cloned into pX458 (pSpCas9(BB)‐2A‐GFP (PX458) was a gift from Feng Zhang (Addgene plasmid # 48138)) (Ran et al , ) vector by single‐step cloning using BbsI restriction sites to generate Gsk3 KO construct. pX458 vector was used as a control (Gsk3 Ctrl construct).

Techniques: Transfection, Western Blot, Expressing, CRISPR, Construct, Immunostaining, Staining

Figure 7. The UFMylation pathway contributes to Toxoplasma virulence (A) Rank-ordered plots for in vivo fitness scores from the endomembrane-nucleus sublibrary (left) and the metabolism sublibrary (right). UFMylation-related genes are marked in red. Error bars (gray) represent SEM. (B) Fitness scores of the UFMylation-related genes in Vero cells (in vitro), WT mice (WT), and Ifngr1/ mice (KO) are shown as box plots. Each dot represents fitness scores for individual gRNAs. ***p < 0.001; *p < 0.05 (Wilcoxon rank-sum test). (C) The conserved C-terminal amino acid sequences of the UFM1 homologs from indicated apicomplexan (blue) and model organisms (black). The conserved glycine residue is shown by the arrow. (D) Survival curves of WT mice with intra-footpad infection of 103 tachyzoites of WT (n = 10), DUFM1 (n = 6), DUFM1 + UFM1 (WT) (n = 7), and DUFM1 + UFM1 (G87A) (n = 7). ***p < 0.001; N.S., not significant (log-rank test). (E) Complementations of FLAG-tagged WT and G87A UFM1 protein in DUFM1 parasites. (F) Survival curves of WT (n = 10) or Ifngr1/ (n = 9) mice with intra-footpad infection of 103 tachyzoites of DUFM1 parasites. ***p < 0.001 (log-rank test). See also Figure S6.

Journal: Cell reports

Article Title: Host genetics highlights IFN-γ-dependent Toxoplasma genes encoding secreted and non-secreted virulence factors in in vivo CRISPR screens.

doi: 10.1016/j.celrep.2023.112592

Figure Lengend Snippet: Figure 7. The UFMylation pathway contributes to Toxoplasma virulence (A) Rank-ordered plots for in vivo fitness scores from the endomembrane-nucleus sublibrary (left) and the metabolism sublibrary (right). UFMylation-related genes are marked in red. Error bars (gray) represent SEM. (B) Fitness scores of the UFMylation-related genes in Vero cells (in vitro), WT mice (WT), and Ifngr1/ mice (KO) are shown as box plots. Each dot represents fitness scores for individual gRNAs. ***p < 0.001; *p < 0.05 (Wilcoxon rank-sum test). (C) The conserved C-terminal amino acid sequences of the UFM1 homologs from indicated apicomplexan (blue) and model organisms (black). The conserved glycine residue is shown by the arrow. (D) Survival curves of WT mice with intra-footpad infection of 103 tachyzoites of WT (n = 10), DUFM1 (n = 6), DUFM1 + UFM1 (WT) (n = 7), and DUFM1 + UFM1 (G87A) (n = 7). ***p < 0.001; N.S., not significant (log-rank test). (E) Complementations of FLAG-tagged WT and G87A UFM1 protein in DUFM1 parasites. (F) Survival curves of WT (n = 10) or Ifngr1/ (n = 9) mice with intra-footpad infection of 103 tachyzoites of DUFM1 parasites. ***p < 0.001 (log-rank test). See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER T. gondii: Strain RH/Dhxgprt/Dku80/DTGGT1_203160 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DGST2 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DEPT1 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DRAB4 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DHMGB This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DALG2 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DDGAT1 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DTGGT1_211695 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DPDX1 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DPDX2 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DEF-P This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DTGGT1_204350 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DTGGT1_215890 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DUSPase This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DRad23 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DHID1 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DSui1 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DGRA17 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DGRA72+ GRA72-HA This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/ GRA23-HA This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DGRA72/GRA23-HA This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DUFM1 This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DUFM1+UFM1(WT) This study N/A T. gondii: Strain RH/Dhxgprt/Dku80/DUFM1+UFM1(G87A) This study N/A C57BL/6 mice SLC N/A Ifngr1 / mice Sasai et al.71 N/A Oligonucleotides For primers and oligonucleotides, see Table S4 This study N/A Recombinant DNA For each gRNA sublibrary, see Tables S1–S3 This study N/A pU6-Universal Sidik et al.72 Addgene plasmid: #52694 pSAG1-GRA72-HA-HXGPRT This study N/A pUPRT-UFM1(WT) This study N/A pUPRT-UFM1(G87A) This study N/A (Continued on next page) Cell Reports 42, 112592, June 27, 2023 17

Techniques: In Vivo, In Vitro, Residue, Infection

( A ) Workflow of the CRISPR screen in retinal pigment epithelium (RPE1) cells, which were transduced with a lentiviral Genome-Scale CRISPR Knock-out (GeCKO) single guide RNA (sgRNA) library and selected for the sgRNA expression and then survival after treatments with the proteasome inhibitor MG132. Individual surviving cell colonies were collected for sequencing and subsequent analysis. ( B ) Left: The cytotoxicity analysis of wild-type (WT) and DBT knockout (KO) RPE1 cells treated with MG132 at different doses for 96 hr (n=3). Right: The time course analysis of MG132-induced cytotoxicity in the WT and DBT KO cells (n=3). ( C ) Immunoblot analysis of WT RPE1, DBT KO, and DBT’ cells. The DBT’ cells expressed an engineered DBT cDNA that resisted DBT-targeted Cas9 cleavage and rescued the DBT expression in the KO cells. ( D ) Cell viability was measured by Calcein-AM staining in WT RPE1, DBT KO, and DBT’ cells treated with MG132 (2 μM, 96 hr). Scale bar, 100 μm. ( E ) Quantification of the cell viability measured by Calcein-AM staining in ( D ) (n=9). ( F ) Left: Immunoblot analysis of RPE1 cells transfected with DBT shRNAs and non-targeting control shRNAs. Right: Quantification of the cell viability under treatment with MG132 (2 μM, 48 hr), as measured by Calcein-AM staining (n=4). ( G ) Immunoblotting and quantification of cleaved PARP as an MG132-induced cell death marker (n=4). ( H ) Immunoblotting and quantification of cleaved Caspase 3 as an MG132-induced cell death marker (n=3). Error bars represent means ± SEM. *p≤0.05; **p≤0.01; ****p≤0.0001. Figure 1—source data 1. Original and uncropped blots for . Figure 1—source data 2. Original and uncropped blots for . Figure 1—source data 3. Original and uncropped blots for . Figure 1—source data 4. Original and uncropped blots for .

Journal: eLife

Article Title: DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment

doi: 10.7554/eLife.91002

Figure Lengend Snippet: ( A ) Workflow of the CRISPR screen in retinal pigment epithelium (RPE1) cells, which were transduced with a lentiviral Genome-Scale CRISPR Knock-out (GeCKO) single guide RNA (sgRNA) library and selected for the sgRNA expression and then survival after treatments with the proteasome inhibitor MG132. Individual surviving cell colonies were collected for sequencing and subsequent analysis. ( B ) Left: The cytotoxicity analysis of wild-type (WT) and DBT knockout (KO) RPE1 cells treated with MG132 at different doses for 96 hr (n=3). Right: The time course analysis of MG132-induced cytotoxicity in the WT and DBT KO cells (n=3). ( C ) Immunoblot analysis of WT RPE1, DBT KO, and DBT’ cells. The DBT’ cells expressed an engineered DBT cDNA that resisted DBT-targeted Cas9 cleavage and rescued the DBT expression in the KO cells. ( D ) Cell viability was measured by Calcein-AM staining in WT RPE1, DBT KO, and DBT’ cells treated with MG132 (2 μM, 96 hr). Scale bar, 100 μm. ( E ) Quantification of the cell viability measured by Calcein-AM staining in ( D ) (n=9). ( F ) Left: Immunoblot analysis of RPE1 cells transfected with DBT shRNAs and non-targeting control shRNAs. Right: Quantification of the cell viability under treatment with MG132 (2 μM, 48 hr), as measured by Calcein-AM staining (n=4). ( G ) Immunoblotting and quantification of cleaved PARP as an MG132-induced cell death marker (n=4). ( H ) Immunoblotting and quantification of cleaved Caspase 3 as an MG132-induced cell death marker (n=3). Error bars represent means ± SEM. *p≤0.05; **p≤0.01; ****p≤0.0001. Figure 1—source data 1. Original and uncropped blots for . Figure 1—source data 2. Original and uncropped blots for . Figure 1—source data 3. Original and uncropped blots for . Figure 1—source data 4. Original and uncropped blots for .

Article Snippet: The specific gRNA sequences were selected by using the CRISPR design tool from Benchling, Inc The gRNAs were cloned into the gRNA/Cas9-expressing vector pLenti-CRISPR v2, conferring resistance to puromycin (Addgene 52961).

Techniques: CRISPR, Transduction, Knock-Out, Expressing, Sequencing, Western Blot, Staining, Transfection, Control, Marker