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1) Product Images from "CRISPR/Cas9-Mediated Genome Editing Corrects Dystrophin Mutation in Skeletal Muscle Stem Cells in a Mouse Model of Muscle Dystrophy"
Article Title: CRISPR/Cas9-Mediated Genome Editing Corrects Dystrophin Mutation in Skeletal Muscle Stem Cells in a Mouse Model of Muscle Dystrophy
Journal: Molecular Therapy. Nucleic Acids
Figure Legend Snippet: Correction of the Dmd Mutation Using ssODN Donor with CRISPR/Cas9 System in Fibrin-Expanded MuSCs (A) Representative images showing delivery of CRISPR/Cas9 components into fibrin-expanded cells. Bulk skeletal muscle cells were cultured in soft 3D fibrin gel. When round-shaped MuSCs became the dominant cell type in soft 3D fibrin gel, cells were transfected with a cocktail of gRNA, ssODN, and pmax-GFP by Lipofectamine 3000. Six hours after transfection, cells were then infected with adenovirus AdV-Cas9-RFP, which co-expresses RFP and CRISPR/Cas9. GFP was used to reflect transfection efficiency of ssODN and gRNA. Scale bar, 100 μm. (B) Genotyping PCR using Mdx-F2 and SM-R2 indicating HDR-mediated Dmd correction in the fibrin-expanded MuSCs (n = 3). P, positive control using synthesized donor DNA fragment; X, genomic DNA from uncorrected mdx muscle cells; FBC, genomic DNA from corrected fibrin-expanded bulk muscle cells from mdx mice; M, 100-bp DNA marker. (C) TseI digestion confirming HDR-mediated Dmd correction in fibrin gel-expanded MuSCs. Allele-specific PCR products amplified by Mdx-F1 and SM-R2 from genomic DNA of expanded MuSC were sub-cloned into TOPO cloning vector, followed by colony-PCR with the same pair of allele-specific primers. The 97-bp PCR products from individual colonies were directly digested by TseI, which was incorporated by ssODN-directed HDR, and resulted in two fragments of 73 and 24 bp, respectively. Only the 73-bp fragment was visible by electrophoresis in 3% agarose gel. (D) Confirmation of HDR-mediated Dmd correction in fibrin-expanded MuSCs by DNA sequencing. TOPO clones referred to in (C) were sequenced. Silent mutations were indicated with green letters. Point mutations were highlighted in red.
Techniques Used: Mutagenesis, CRISPR, Cell Culture, Transfection, Infection, Polymerase Chain Reaction, Positive Control, Synthesized, Mouse Assay, Marker, Amplification, Clone Assay, Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, DNA Sequencing
Figure Legend Snippet: Correction of Dmd Gene in Soft 3D Fibrin-Expanded MuSCs Using Adenoviral Vector Delivery of RNA-Guided CRISPR/Cas9 and Donor DNA (A) Diagram of adenoviral vectors expressing Cas9 (AdV-Cas9) and harboring gRNA expression cassette and Dmd -specific donor template. The mdx-gRNA2 shown in Figure 2 B was used. The donor template was a 1,314-bp DNA fragment with 591- and 722-bp homology arms flanking the mutation site. The silent mutations constituting TseI restriction enzyme site in ssODN were also incorporated in this longer donor template. (B) Scheme of adult MuSC-based gene therapy for DMD in mdx mice. Bulk skeletal muscle cells were isolated from mdx mice and then cultured in soft 3D fibrin gel. When morphologically round MuSCs became evident (3∼4 days), CRISPR/Cas9 and donor DNA complexes were delivered to initiate targeted genome editing for correcting Dmd mutations. Cells were allowed to expand in fibrin gel for 3 more days to propagate Dmd-corrected MuSCs. Expanded MuSCs were then transplanted in mdx mice. (C) Representative images showing adenoviral delivery of CRISPR/Cas9 components into fibrin-expanded cells. Bulk skeletal muscle cells were cultured in soft 3D fibrin gel. When round MuSCs became the dominant cell type in soft 3D fibrin gel, cells were coinfected with adenoviruses expressing CRISPR/Cas9 (red) and carrying gRNA and donor DNA (green). RFP was used to track infection efficiency of adenovirus expressing Cas9, whereas GFP was used to reflect transfection efficiency of donor DNA and gRNA. Scale bar, 100 μm. (D) Schematic diagram for assessing correction of Dmd mutation by adenoviral delivery of CRISPR/Cas9 complexes. To avoid interference of donor DNA as template in PCR, genome DNA was amplified first by a pair of primers (Mdx-F1 and Mdx-R1) residing outside of donor DNA sequence. The resultant PCR products containing both original and mdx -corrected fragments were then purified and subcloned into TOPO-TA cloning vector. After transformation, single bacterial colonies were picked up for allele-specific PCR using primers of LA-F and SM-R2 to screen for the corrected clones. To confirm allele-specific PCR results, colony PCR was further performed using primers of Mdx-F2 and SM-R1, and the resultant product was subjected to TseI digestion. TseI site was designed and introduced in the donor DNA. (E) Genotyping result of HDR-mediated Dmd correction in the fibrin-expanded MuSCs (n = 3 independent experiments). M, 100-bp DNA marker; D, donor DNA plasmids; X, genomic DNA from uncorrected mdx muscle cells; C, genomic DNA from corrected fibrin-expanded mdx muscle cells. Mdx-F1 and Mdx-R1 were used for the first round of PCR, and LA-F and SM-R2 were used for the second round of PCR. (F) TseI digestion confirming HDR-mediated Dmd correction in fibrin gel-expanded MuSCs. DNA fragments (lane C, left panel of D) from first round of PCR were sub-cloned into TOPO-TA cloning vector, followed by colony-PCR with primers of Mdx-F2 and SM-R1. PCR products from individual colonies were directly digested by TseI.
Techniques Used: Plasmid Preparation, CRISPR, Expressing, Mutagenesis, Mouse Assay, Isolation, Cell Culture, Infection, Transfection, Polymerase Chain Reaction, Amplification, Sequencing, Purification, TA Cloning, Transformation Assay, Clone Assay, Marker
Figure Legend Snippet: Design of gRNAs and ssODN for CRISPR/Cas9-Mediated Gene Editing (A) Design of ssODN as HDR DNA template. ssODN, which contains 41 bp of homology arms flanking each side of the mutation site and targets the non-transcribed strand of Dmd gene. ssODN was phosphorothioate-modified at both ends (denoted with * [refer to Table S1 ]) and incorporated four silent mutations (green), which prevent binding of gRNA2/Cas9 and add a TseI restriction enzyme site for genotyping and verification of HDR-mediated gene correction. (B) Schematic of gRNAs targeting sequences of Dmd (blue) and PAM sequence (red). Red arrowhead indicates the cleavage site by CRISPR/Cas9. gRNA1 and gRNA2 were the two gRNAs specific for Dmd . (C) PCR validation of CRISPR/Cas9-mediated DNA cleavage of genomic DNA target. Mouse muscle-derived fibroblasts were transfected with CRISPR/Cas9, gRNA1, and gRNA2 by Lipofectamine 3000. CRISPR/Cas9 cut at each of the two gRNA-targeted sequences in Dmd , which resulted in 47-bp shorter PCR products when amplified with two primers of Mdx-F2 and Mdx-R2 (see Table S1 ), which locate in 5′ end of gRNA2 site and 3′ ends of gRNA1 site, respectively.
Techniques Used: CRISPR, Mutagenesis, Modification, Binding Assay, Sequencing, Polymerase Chain Reaction, Derivative Assay, Transfection, Amplification