Journal: Bio-protocol

Article Title: Construction of Viral Vectors for Cell Type-specific CRISPR Gene Editing in the Adult Mouse Brain

doi: 10.21769/BioProtoc.3334

Figure Lengend Snippet: Our method consists of four steps: (1) crossing Cre-dependent spCas9 knockin mice with a Cre-driver mouse line to express spCas9 in the target neural populations, (2) cloning of sgRNA into the spCas9 expressing plasmid, (3) construction of an AAV vector tandemly expressing dual sgRNA, and (4) AAV packaging and stereotaxic viral injection into the target brain area.

Article Snippet: Pipette tips 24-well plate (coated) (Thermo Fisher Scientific, catalog number: 142475) 1 ml plastic tube NEB10-beta Competent E. coli (New England Biolabs, catalog number: C3019) pX458 (Addgene, catalog number: 48138) pAAV EF1α DIO mCherry (Addgene, catalog number: 20299) BbsI-HF (New England Biolabs, catalog number: R3539) MluI-HF (New England Biolabs, catalog number: R3198) DpnI (New England Biolabs, catalog number: R0176) Alkaline Phosphatase, Calf Intestinal (CIP) (New England Biolabs, catalog number: M0290) Guide oligonucleotides (see Procedure A) Quick ligation kit (New England Biolabs, catalog number: M2200) Gibson Assembly Master Mix (New England Biolabs, catalog number: E2611) PrimeSTAR HS DNA polymerase (Takara, catalog number: R010) dNTP Mix 10 mM each (the mix of 10 mM of dTTP, dCTP, dGTP and dATP) (GenScript, catalog number: {"type":"entrez-nucleotide","attrs":{"text":"C01689","term_id":"1433919","term_text":"C01689"}} C01689 ) QIAquick Gel Extraction kit (QIAGEN, catalog number: 28704) QIAprep Spin Miniprep kit (QIAGEN, catalog number: 27104) Tris-EDTA, (1x Solution) (Fisher BioReagents, catalog number: BP2473) LB Broth EZMix Powder (Sigma, catalog number: L7658) LB Agar (Fisher BioReagents, catalog number: BP1425) Ampicillin (Gemini Bio-Products, catalog number: 400-130P) Primer to verify the cloning of sgRNA into pX458: 5’-gactatcatatgcttaccgt-3’ Primer to verify the tandem cloning of sgRNAs into pAAV: 5’-actgacgggcaccggagcca-3’ Primers for Gibson Assembly: Primer S1: 5’-taggggttcctgcggccgcacgcgtgagggcctatttc-3’ Primer AS1: 5’-ataggccctctctagaaaaaaagcaccgactc-3’ Primer S2: 5’-tttttctagagagggcctatttcccatg-3’ Primer AS2: 5’-atccatctttgcaaagcttacgcgtaaaaaagcaccgac-3’ Optional (see Procedure C) NIH3T3 cell (ATCC, catalog number: CRL-1658) AAV vector (pX458-sgRNA or pX458 empty as negative control ) Primers: You can design the primers to amplify the target exons by CHOPCHOP (see Procedure C) PrimeSTAR HS DNA polymerase FuGENE6 Transfection Reagent (Promega, catalog number: E2693) DMEM (1x) (Gibco, catalog number: 11995-065) Fetal Bovine Serum (Corning, catalog number: 35-015-CV) Penicillin/Streptomycin (Gibco, catalog number: 15070-063) PBS, pH 7.4 (Gibco, catalog number: 10010-023) TrypLE Select (1x) (Gibco, catalog number: 12563-011) QuickExtract DNA Extraction Solution (Epicentre, catalog number: QE0905T) T7 Endonuclease I (New England Biolabs, catalog number: M0302)

Techniques: Knock-In, Clone Assay, Expressing, Plasmid Preparation, Injection

Journal: Bio-protocol

Article Title: Construction of Viral Vectors for Cell Type-specific CRISPR Gene Editing in the Adult Mouse Brain

doi: 10.21769/BioProtoc.3334

Figure Lengend Snippet: A. Cloning of guide oligonucleotides. Oligonucleotides with 20-nt guide sequence and overhangs for cloning into BbsI site in pX458 were annealed and cloned into BbsI-digested pX458. B. Cloning of U6 promoter-driven sgRNAs. U6 promoter followed by sgRNA1 or sgRNA2 were PCR amplified, respectively, by using primers with overhangs for Gibson Assembly. C. Gibson Assembly of the dual sgRNA virus vector. U6-gRNA1 and U6-gRNA2 were cloned into MluI-digested pAAV EF1α DIO mCherry using Gibson Assembly.

Article Snippet: Pipette tips 24-well plate (coated) (Thermo Fisher Scientific, catalog number: 142475) 1 ml plastic tube NEB10-beta Competent E. coli (New England Biolabs, catalog number: C3019) pX458 (Addgene, catalog number: 48138) pAAV EF1α DIO mCherry (Addgene, catalog number: 20299) BbsI-HF (New England Biolabs, catalog number: R3539) MluI-HF (New England Biolabs, catalog number: R3198) DpnI (New England Biolabs, catalog number: R0176) Alkaline Phosphatase, Calf Intestinal (CIP) (New England Biolabs, catalog number: M0290) Guide oligonucleotides (see Procedure A) Quick ligation kit (New England Biolabs, catalog number: M2200) Gibson Assembly Master Mix (New England Biolabs, catalog number: E2611) PrimeSTAR HS DNA polymerase (Takara, catalog number: R010) dNTP Mix 10 mM each (the mix of 10 mM of dTTP, dCTP, dGTP and dATP) (GenScript, catalog number: {"type":"entrez-nucleotide","attrs":{"text":"C01689","term_id":"1433919","term_text":"C01689"}} C01689 ) QIAquick Gel Extraction kit (QIAGEN, catalog number: 28704) QIAprep Spin Miniprep kit (QIAGEN, catalog number: 27104) Tris-EDTA, (1x Solution) (Fisher BioReagents, catalog number: BP2473) LB Broth EZMix Powder (Sigma, catalog number: L7658) LB Agar (Fisher BioReagents, catalog number: BP1425) Ampicillin (Gemini Bio-Products, catalog number: 400-130P) Primer to verify the cloning of sgRNA into pX458: 5’-gactatcatatgcttaccgt-3’ Primer to verify the tandem cloning of sgRNAs into pAAV: 5’-actgacgggcaccggagcca-3’ Primers for Gibson Assembly: Primer S1: 5’-taggggttcctgcggccgcacgcgtgagggcctatttc-3’ Primer AS1: 5’-ataggccctctctagaaaaaaagcaccgactc-3’ Primer S2: 5’-tttttctagagagggcctatttcccatg-3’ Primer AS2: 5’-atccatctttgcaaagcttacgcgtaaaaaagcaccgac-3’ Optional (see Procedure C) NIH3T3 cell (ATCC, catalog number: CRL-1658) AAV vector (pX458-sgRNA or pX458 empty as negative control ) Primers: You can design the primers to amplify the target exons by CHOPCHOP (see Procedure C) PrimeSTAR HS DNA polymerase FuGENE6 Transfection Reagent (Promega, catalog number: E2693) DMEM (1x) (Gibco, catalog number: 11995-065) Fetal Bovine Serum (Corning, catalog number: 35-015-CV) Penicillin/Streptomycin (Gibco, catalog number: 15070-063) PBS, pH 7.4 (Gibco, catalog number: 10010-023) TrypLE Select (1x) (Gibco, catalog number: 12563-011) QuickExtract DNA Extraction Solution (Epicentre, catalog number: QE0905T) T7 Endonuclease I (New England Biolabs, catalog number: M0302)

Techniques: Clone Assay, Sequencing, Amplification, Plasmid Preparation

Journal: Cytotechnology

Article Title: Generation of PTEN knockout bone marrow mesenchymal stem cell lines by CRISPR/Cas9-mediated genome editing

doi: 10.1007/s10616-017-0183-3

Figure Lengend Snippet: PTEN-KO validation of the MSCs. a The designed target site exon region. b The map of empty vector (from Addgene, Massachusetts, USA). c T7E1 digestion products were analyzed by agarose gel electrophoresis. The pSpCas9(BB)-2A-GFP-T2 (px458-T2) plasmid showed the highest Knock-out performance. d DNA was extracted from plasmid-transfected BMSCs and amplified through PCR. The PTEN bands did not exist after transfection e Western blot confirmed PTEN was knocked out

Article Snippet: All PCR products were verified by DNA sequencing, namely pSpCas9(BB)-2A-GFP-T1 (px458-T1), pSpCas9(BB)-2A-GFP (px458-T2) and pSpCas9(BB)-2A-GFP-T3 (px458-T3). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1 caption a7 PTEN-KO validation of the MSCs. a The designed target site exon region. b The map of empty vector (from Addgene, Massachusetts, USA). c T7E1 digestion products were analyzed by agarose gel electrophoresis.

Techniques: Biomarker Discovery, Plasmid Preparation, Agarose Gel Electrophoresis, Knock-Out, Transfection, Amplification, Western Blot

Journal: Cell Death and Differentiation

Article Title: mTORC1-independent autophagy regulates receptor tyrosine kinase phosphorylation in colorectal cancer cells via an mTORC2-mediated mechanism

doi: 10.1038/cdd.2017.41

Figure Lengend Snippet: Basal autophagy-mediated regulation of c-MET phosphorylation involves mTORC2 signalling. (a) Immunoblot depicts levels of Rictor protein expression in HCT-116 cells treated with Scramble (Scr) and Rictor siRNA for 5 days. β-actin was used as loading control. Bar plots represent densitometric quantification of Rictor expression levels normalised to β-actin. The bars represent mean±S.D. of n=3; **P<0.01. Unpaired Student's t-test statistical analysis was used. (b) Immunoblot depicts reduced phosphorylation levels of c-MET and AKT but not S6 ribosomal protein following Rictor siRNA for 5 days. β-actin was used as loading control. Bar plots represent densitometric quantification of p-c-MET Y1234/1235, pAKT S473 and pS6 ribosomal protein relative to total c-MET, total AKT and total S6 ribosomal protein, respectively. The bars represent mean±S.D. of n=4; ns=non-statistically significant; *P<0.05 and ***P<0.001. Unpaired Student's t-test statistical analysis was used. (c) Rictor knockdown does not affect levels of autophagy. Five days after Scr and Rictor-siRNA delivery, HCT-116 cells were treated ±10 μM CQ for 6 h and total cell lysates were analysed using immunoblot for LC3B protein. β-actin was used as loading control. Bar plots represent densitometric analysis of LC3-II/LC3-I levels. The bars represent mean±S.D. of n=4; ns=non-statistically significant. Two-way ANOVA statistical analysis was used. (d) Immunoblot depicts levels of Rictor protein expression in autophagy-proficient (gRNA Ctrl) and deficient (gRNA ATG7) HCT-116 cells transfected with Rictor gRNA. β-actin was used as loading control. Bar plots represent densitometric quantification of Rictor expression levels normalised to β-actin. The bars represent mean±S.D. of n=4; ns-non-statistically significant; *P<0.05; **P<0.01. Two-way ANOVA statistical analysis was used. (e) Immunoblot depicts reduced phosphorylation levels of c-MET but not pS6 ribosomal protein following Rictor gRNA transfection. β-actin was used as loading control. Bar plots represent densitometric quantification of p-c-MET Y1234/1235 and pS6 ribosomal protein relative to total c-MET and total S6 ribosomal protein, respectively. The bars represent mean±S.D. of n=4; ns=non-statistically significant; **P<0.01. Two-way ANOVA statistical analysis was used. (f) Downregulation of Rictor does not affect c-MET internalisation nor its accumulation in intracellular LC3B-positive structures. Representative images from confocal sections of autophagy-proficient Rictor WT (Ctrl) or KO (Ctrl_Rictor gRNA) HCT-116 cells treated with 10 μM CQ for 6 h. Cells were fixed and stained for total c-MET (green), LC3B (red) and nucleus (Hoechst dye-blue). Scale bar corresponds to 5 μm. Orthogonal reconstruction of serial confocal slices is shown (Y–Z and X–Z axis with 1: merge of total c-MET/LC3B/Hoechst; 2: total c-MET; 3: LC3B). Yellow lines on the confocal section indicate from where the orthogonal views were built. (g–h) mTOR kinase inhibition in autophagy-proficient cells reduces phosphorylation levels of c-MET without affecting autophagic flux. Autophagy-proficient (shEGFP) and compromised (shATG7 E8) HCT-116 KRAS WT cells were treated with 10 ng/ml Dox for 5 days. At day 5 of Dox treatment, cells were treated with 2 nM of the Torin2 inhibitor ±10 μM CQ for 6 h and total cell lysates were subjected to immunoblotting for the indicated proteins. Bar plots represent densitometric quantification of p-c-MET Y1234/1235 relative to total c-MET protein and pAKT S473 relative to total AKT protein (g) and LC3-II/LC3-I levels (h). The bars represent mean±S.D. of n=4 (g) and n=3 (h); ns=non-significant; ***P<0.001. Two-way ANOVA statistical analysis was used

Article Snippet: Autophagy-proficient and deficient HCT-116 cells were transiently transfected either with pSpCas9BB-2 A-GFP (PX458) empty vector or -Rictor CRISPR guide RNA (gRNA) 3 expressing vector by using FuGENE transfection reagent (#E2311, Promega Corp., Madison, WI, USA), in accordance with the instructions of the manufacturer.

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Knockdown, Transfection, Staining, Inhibition