sgrna (tracrrna and crrna) with ms2 stem loop fragment  (Addgene inc)

Journal: Molecular Therapy. Nucleic Acids

Article Title: One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes

doi: 10.1016/j.omtn.2017.06.007

Figure Lengend Snippet: Identification of the Best sgRNAs in Gene Activation by 293FT-SAM (A) PB vectors used for creating 293FT-SAM. (B) Generation of 293FT-SAM cell line by co-transfecting 293FT with PB-SAM and PBase expression vectors. qPCR results showed that SAM components MS2-p65-HSF1 (MSPH) and dCas9-VP64 were stably expressed for at least 90 days. (C–E) qPCR results of gene expression activation of individual TFs (ASCL1, NEUROG1, NEUROG2, OLIG2, SOX10, LHX3, NKX2-2, MNX1, MYT1, FOXG1, LHX2, SOX8, OLIG1, SOX2, ISL1, SOX11, SOX9, POU3F2, NFIA, NFIB, and NFIX) and lncRNAs (RMST, HAR1B, and HAR1A) after transfecting 293FT-SAM cell line with corresponding sgRNA vectors. A total of three sgRNAs per gene was tested separately to evaluate their activation efficiency. The rightmost bars for each gene represent the extent of gene activation when all three sgRNA vectors for that gene were co-transfected. (F) A pie chart showing the range of fold change of gene expression after each sgRNA vector was transfected into the 293FT-SAM line. For each gene, three sgRNAs were tested separately. A total of 72 sgRNAs was tested. Basically, 43 sgRNAs were able to augment target gene expression by >2-fold. (G) For the 24 genes targeted, 19 genes were activated to be expressed at >2-fold. The qPCR results were normalized to GAPDH mRNA level. P2A, self-cleaving peptide P2A sequence; Hygro, hygromycin resistance cassette; Blast, blasticidin resistance cassette; NLS, nuclear localization signal. In (B)–(E), data are presented as mean ± SEM (n = 3).

Article Snippet: The sgRNA (tracrRNA and crRNA) with MS2 stem loop fragment ( A) (Addgene, 61427) was digested and cloned into the PB vector via Gibson assembly.

Techniques: Activation Assay, Expressing, Stable Transfection, Gene Expression, Transfection, Plasmid Preparation, Targeted Gene Expression, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes

doi: 10.1016/j.omtn.2017.06.007

Figure Lengend Snippet: A Schematic Representation of Constructing PB-CRISPRa All-in-One Vectors with Multisite Gateway Cloning Strategy The PB-SAM vector was amplified to become PB-SAM R1-R2 DEST vector containing attR1 and attR2 sites, a ccdB cassette, and a chloramphenicol resistance cassette (CmR). PB-sgRNA (MS2) vectors containing different sgRNA inserts were amplified to be attached to appropriate attB sites. All-in-one vectors were assembled by four-way LR reactions. P2A, self-cleaving peptide P2A sequence; Hygro, hygromycin resistance cassette; Blast, blasticidin resistance cassette; Amp R , ampicillin resistance cassette; Kan R , kanamycin resistance cassette; NLS, nuclear localization signal; ITR, inverted terminal repeat sequence.

Article Snippet: The sgRNA (tracrRNA and crRNA) with MS2 stem loop fragment ( A) (Addgene, 61427) was digested and cloned into the PB vector via Gibson assembly.

Techniques: Cloning, Plasmid Preparation, Amplification, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes

doi: 10.1016/j.omtn.2017.06.007

Figure Lengend Snippet: Identification of the Best sgRNAs in Gene Activation by 293FT-SAM (A) PB vectors used for creating 293FT-SAM. (B) Generation of 293FT-SAM cell line by co-transfecting 293FT with PB-SAM and PBase expression vectors. qPCR results showed that SAM components MS2-p65-HSF1 (MSPH) and dCas9-VP64 were stably expressed for at least 90 days. (C–E) qPCR results of gene expression activation of individual TFs (ASCL1, NEUROG1, NEUROG2, OLIG2, SOX10, LHX3, NKX2-2, MNX1, MYT1, FOXG1, LHX2, SOX8, OLIG1, SOX2, ISL1, SOX11, SOX9, POU3F2, NFIA, NFIB, and NFIX) and lncRNAs (RMST, HAR1B, and HAR1A) after transfecting 293FT-SAM cell line with corresponding sgRNA vectors. A total of three sgRNAs per gene was tested separately to evaluate their activation efficiency. The rightmost bars for each gene represent the extent of gene activation when all three sgRNA vectors for that gene were co-transfected. (F) A pie chart showing the range of fold change of gene expression after each sgRNA vector was transfected into the 293FT-SAM line. For each gene, three sgRNAs were tested separately. A total of 72 sgRNAs was tested. Basically, 43 sgRNAs were able to augment target gene expression by >2-fold. (G) For the 24 genes targeted, 19 genes were activated to be expressed at >2-fold. The qPCR results were normalized to GAPDH mRNA level. P2A, self-cleaving peptide P2A sequence; Hygro, hygromycin resistance cassette; Blast, blasticidin resistance cassette; NLS, nuclear localization signal. In (B)–(E), data are presented as mean ± SEM (n = 3).

Article Snippet: The sgRNA (tracrRNA and crRNA) with MS2 stem loop fragment ( A) (Addgene, 61427) was digested and cloned into the PB vector via Gibson assembly.

Techniques: Activation Assay, Expressing, Stable Transfection, Gene Expression, Transfection, Plasmid Preparation, Targeted Gene Expression, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes

doi: 10.1016/j.omtn.2017.06.007

Figure Lengend Snippet: A Schematic Representation of Constructing PB-CRISPRa All-in-One Vectors with Multisite Gateway Cloning Strategy The PB-SAM vector was amplified to become PB-SAM R1-R2 DEST vector containing attR1 and attR2 sites, a ccdB cassette, and a chloramphenicol resistance cassette (CmR). PB-sgRNA (MS2) vectors containing different sgRNA inserts were amplified to be attached to appropriate attB sites. All-in-one vectors were assembled by four-way LR reactions. P2A, self-cleaving peptide P2A sequence; Hygro, hygromycin resistance cassette; Blast, blasticidin resistance cassette; Amp R , ampicillin resistance cassette; Kan R , kanamycin resistance cassette; NLS, nuclear localization signal; ITR, inverted terminal repeat sequence.

Article Snippet: The sgRNA (tracrRNA and crRNA) with MS2 stem loop fragment ( A) (Addgene, 61427) was digested and cloned into the PB vector via Gibson assembly.

Techniques: Cloning, Plasmid Preparation, Amplification, Sequencing