pac sgrna cas9 vector  (Addgene inc)

Journal: MethodsX

Article Title: CRISPR-editing of the virus vector Aedes albopictus cell line C6/36, illustrated by prohibitin 2 gene knockout

doi: 10.1016/j.mex.2024.102817

Figure Lengend Snippet: Cloning of the CRISPR RNA oligonucleotides to create single guide RNA. (a) (top) Plasmid map of pAc-sgRNA-Cas9 showing the BspQI cloning sites and the guide RNA (gRNA)-scaffold under the dU6–2 promoter and the Cas9 under the Ac5 promoter. The plasmid contains ampicillin- (AmpR) and puromycin-resistant (PuroR) genes. The plasmid map was created by SnapGene Viewer. (middle) Part of the plasmid map is enlarged to show the sequences of the dU6–2 promoter, the BspQ1 restriction enzyme sites and the gRNA scaffold. The trans-activating CRISPR RNA (tracrRNA) oligonucleotides AlbopPHB2sgRNAF and AlbopPHB2sgRNAR to be inserted into the vector are displayed underneath and boxed in red. (bottom) Chromatogram showing the correct insertion of the tracrRNA sequence (boxed in red) into the vector to create single guide RNA (sgRNA). (b) Schematic of the cloning procedures. (left) the plasmid vector was digested with BspQ1 and then dephosphorylated. (right) The forward and reverse oligonucleotides were annealed by heating at 95 °C and then cooling to <30 °C. The annealed oligonucleotides were phosphorylated and ligated into the dephosphorylated vector. The ligation mix was transformed into competent Escherichia coli cells and colonies were screened by PCR. Plasmid DNA was extracted from positive clones and sequenced.

Article Snippet: Two micrograms of the plasmid vector pAc-sgRNA-Cas9 (Addgene #49330) ( a) were digested with 0.4 U/µl of BspQ1 (New England Biolabs) in a final volume of 50µl and dephosphorylated by 1 unit of Anza™ Alkaline Phosphatase (Invitrogen) at 37 °C for 10 min followed by heat inactivation of the phosphatase at 80 °C for 5 min. Two microliters of the 10x diluted annealed oligonucleotides were ligated to 1.7 µl of the BspQ1-digested vector in Anza™ T4 DNA Ligase Master Mix (Invitrogen) in a final volume of 10 µl at RT for 1 h ( b).

Techniques: Clone Assay, CRISPR, Plasmid Preparation, Sequencing, Ligation, Transformation Assay

Journal: MethodsX

Article Title: CRISPR-editing of the virus vector Aedes albopictus cell line C6/36, illustrated by prohibitin 2 gene knockout

doi: 10.1016/j.mex.2024.102817

Figure Lengend Snippet: Selection of CRISPR-edited cells. (a) Schematic of the selection procedures. (i) Plasmid containing the single guide RNA (sgRNA) or the empty vector control was transfected into C6/36 cells for 48 h. (ii) Transfected cells were seeded at 1 cell per well in a 96-well plate by limiting dilution and then expanded into 24- and then 6-well plates. (iii) Transfected cells were re-seeded at low densities in 100 mm dishes and then selected by 2µg/ml puromycin for 2 weeks. Puromycin was then removed and cells were cultured until colonies appeared. Cell colonies were either (iv) ring-cloned and then seeded at 1 cell per well in a 96-well plate by limiting dilution and then expanded into 24- and then 6-well plates; or (v) ring-cloned and transferred directly into 24-well plate and then expanded into 6-well plate. When cells became confluent in 6-well plate, (vi) half was maintained in cell passage until confirmation of insertions and deletions (INDELs) before expanding to make cell stocks; (vii) half was harvested for genomic DNA (gDNA) extraction for PCR using primer pair IF and IR . The PCR products were resolved by agarose gel electrophoresis to identify the INDELs. An example of an agarose gel showing INDELs is shown. (viii) Mutant clones with INDELs were further sequenced. An example of a chromatogram with mixed sequences is shown. The target sequence is boxed in red and the DNA site cleaved by Cas9 is denoted by a blue scissor. (b) Sequence alignment of the parental and edited PHB2 genes showing a repertoire of INDELs predicted by decomposition of mixed sequences using CRISPR-ID. The target sequence is in red and the protospacer adjacent motif (PAM) in blue. The DNA site cleaved by Cas9 is denoted by a blue scissor.

Article Snippet: Two micrograms of the plasmid vector pAc-sgRNA-Cas9 (Addgene #49330) ( a) were digested with 0.4 U/µl of BspQ1 (New England Biolabs) in a final volume of 50µl and dephosphorylated by 1 unit of Anza™ Alkaline Phosphatase (Invitrogen) at 37 °C for 10 min followed by heat inactivation of the phosphatase at 80 °C for 5 min. Two microliters of the 10x diluted annealed oligonucleotides were ligated to 1.7 µl of the BspQ1-digested vector in Anza™ T4 DNA Ligase Master Mix (Invitrogen) in a final volume of 10 µl at RT for 1 h ( b).

Techniques: Selection, CRISPR, Plasmid Preparation, Control, Transfection, Cell Culture, Clone Assay, Extraction, Agarose Gel Electrophoresis, Mutagenesis, Sequencing

Journal: MethodsX

Article Title: CRISPR-editing of the virus vector Aedes albopictus cell line C6/36, illustrated by prohibitin 2 gene knockout

doi: 10.1016/j.mex.2024.102817

Figure Lengend Snippet: CRISPR gene editing changes PHB2 DNA sequences giving rise to frameshifted and truncated, non-functional proteins. (a) TIDE prediction of insertions and deletions (INDELs) in the mutant clone, SW3. (b) (left panel) Chromatograms showing the target sequence and protospacer adjacent motif (PAM) in the parental sequence of the prohibitin 2 (PHB2) gene and the INDELs in the two cloned allelic gene fragments from the CRISPR-edited mutant cell clone, SW3. Red arrow indicates the position where the DNA was cleaved by Cas9 and INDELs (in red box and alphabets) occurred. Frameshift is highlighted in red single-letter amino acid code above the chromatogram. (Right panel) Alignment of translated amino acid sequences of the PHB2 gene from the parental cells and the two cloned allelic gene fragments from the CRISPR-edited mutant cell clone, SW3. Red indicates frameshift and truncation. The PHB2 domains are delineated above the amino acid sequence and are colour-coded: transmembrane (TM) in green, PHB in blue, coiled-coil (CC) in brown and unconventional mitochondrial targeting sequence (MTS) in pink. The nuclear import signal (NIS) within the PHB domain is in bold. (c) TIDE prediction of INDELs in the two sub-clones, SW3.1 and SW3.2, isolated by re-cloning of the mutant clone, SW3.

Article Snippet: Two micrograms of the plasmid vector pAc-sgRNA-Cas9 (Addgene #49330) ( a) were digested with 0.4 U/µl of BspQ1 (New England Biolabs) in a final volume of 50µl and dephosphorylated by 1 unit of Anza™ Alkaline Phosphatase (Invitrogen) at 37 °C for 10 min followed by heat inactivation of the phosphatase at 80 °C for 5 min. Two microliters of the 10x diluted annealed oligonucleotides were ligated to 1.7 µl of the BspQ1-digested vector in Anza™ T4 DNA Ligase Master Mix (Invitrogen) in a final volume of 10 µl at RT for 1 h ( b).

Techniques: CRISPR, Functional Assay, Mutagenesis, Sequencing, Clone Assay, Isolation

Journal: MethodsX

Article Title: CRISPR-editing of the virus vector Aedes albopictus cell line C6/36, illustrated by prohibitin 2 gene knockout

doi: 10.1016/j.mex.2024.102817

Figure Lengend Snippet:

Article Snippet: Two micrograms of the plasmid vector pAc-sgRNA-Cas9 (Addgene #49330) ( a) were digested with 0.4 U/µl of BspQ1 (New England Biolabs) in a final volume of 50µl and dephosphorylated by 1 unit of Anza™ Alkaline Phosphatase (Invitrogen) at 37 °C for 10 min followed by heat inactivation of the phosphatase at 80 °C for 5 min. Two microliters of the 10x diluted annealed oligonucleotides were ligated to 1.7 µl of the BspQ1-digested vector in Anza™ T4 DNA Ligase Master Mix (Invitrogen) in a final volume of 10 µl at RT for 1 h ( b).

Techniques: CRISPR

Journal: MethodsX

Article Title: CRISPR-editing of the virus vector Aedes albopictus cell line C6/36, illustrated by prohibitin 2 gene knockout

doi: 10.1016/j.mex.2024.102817

Figure Lengend Snippet:

Article Snippet: The insect plasmid vector pAc-sgRNA-Cas9 (Addgene #49330) contains the BspQ1 cloning sites for tracrRNA downstream of the dU6–2 promoter and Cas9 downstream of the actin-5c promoter ( a).

Techniques: CRISPR