Journal: Genome Medicine

Article Title: In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

doi: 10.1186/s13073-021-00876-0

Figure Lengend Snippet: Restoration of dystrophin expression by CRISPR/Cas9. a CRISPR/Cas9-mediated gene editing restored dystrophin mRNA level in myotubes differentiated from DMD–MDSCs. Illustration of primer binding sites (black arrow) for RT-PCR (top row); all detected bands were of the expected size; red arrows indicate bands that were verified by Sanger sequencing in b . M, marker; Un, unedited. b The successful reframing of cDNA in the DMD–MDSCs subjected to three different gene-editing strategies was confirmed by Sanger sequencing. c Western blot analysis of dystrophin expression in targeted (∆45–55, ∆46–54, and INDEL50) or untargeted myotubes differentiated from the DMD–MDSCs; WT MDSCs served as a positive control. Expected molecular weights were 427 kDa for WT and INDEL50, 361 kDa for ∆45–55, and 375 kDa for ∆46–54; MHC served as a loading control. d Representative images of MHC (green) and dystrophin (red, white arrow) expression in targeted (∆45–55, ∆46–54, and INDEL50) and untargeted myotubes differentiated from the DMD–MDSCs as determined by immunocytochemistry; WT MDSCs served as a positive control, and nuclei were stained with DAPI (blue). Scale bar, 100 μm

Article Snippet: CRISPR AAV vectors were generated by PackGene Biotech Co. (Guangzhou, China).

Techniques: Expressing, CRISPR, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Marker, Western Blot, Positive Control, Control, Immunocytochemistry, Staining

Journal: Genome Medicine

Article Title: In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

doi: 10.1186/s13073-021-00876-0

Figure Lengend Snippet: CRISPR/Cas12a -induced rescue of dystrophin expression. a PCR analysis of dystrophin expression in DMD–MDSCs targeted by Cas9/gRNA or Cas12a/gRNA specific to introns 45 and 54, respectively. All detected bands were of the expected size; the band indicated by a red arrow was verified by Sanger sequencing, which confirmed the junction of segmental introns 45 and 54. M, marker. b RT-PCR analysis of dystrophin expression in myotubes differentiated from the DMD–MDSCs targeted by Cas9/gRNA or Cas12a/gRNA, as shown in a . All detected bands were of the expected size; the band indicated by a red arrow was verified by Sanger sequencing, which confirmed the junction of exons 45 and 55. M, marker. c The editing efficiency of three large-scale excision strategies indicated by ddPCR assays. d Nanopore sequencing reads from edited MDSCs mapped at the spliced DMD genome; cut sites were marked by red lines. E, exon. e The base mutation percentage around the cut sites (40 bp for ∆45–55/Cas9, 65 bp for ∆46–54/Cas9 and ∆46–54/Cas12a). f Western blot analysis of dystrophin expression in targeted (∆46–54/Cas9 and ∆46–54/Cas12a) or untargeted myotubes differentiated from the DMD–MDSCs; WT MDSCs served as a positive control. Expected molecular weights were 427 kDa for WT and 375 kDa for Cas9/∆46–54 and Cas12a/∆46–54. MHC served as a loading control. g Representative images of MHC (green) and dystrophin (red, white arrow) expression in Cas12a/∆46–54-targeted myotubes differentiated from the DMD–MDSCs as determined by immunocytochemistry; nuclei were stained with DAPI (blue). Scale bar, 100 μm. h Representative box plots of the efficiency of the different gene-editing strategies in vitro as indicated by the ratio of the number of dystrophin-positive fibers (dystrophin + ) to that of MHC-positive fibers (MHC + ); ∆46–54/Cas9, ∆46–54/ Cas12a, and ∆45–55/Cas9 showed higher efficacy than INDEL50/Cas9 ( P < 0.05, n = 10)

Article Snippet: CRISPR AAV vectors were generated by PackGene Biotech Co. (Guangzhou, China).

Techniques: CRISPR, Expressing, Sequencing, Marker, Reverse Transcription Polymerase Chain Reaction, Nanopore Sequencing, Mutagenesis, Western Blot, Positive Control, Control, Immunocytochemistry, Staining, In Vitro

Journal: Genome Medicine

Article Title: In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

doi: 10.1186/s13073-021-00876-0

Figure Lengend Snippet: In vivo editing of human DMD gene restores dystrophin expression and localization in muscle fibers. (A) Immunofluorescence detection of human dystrophin (green signal in the sarcolemma, white arrow), human lamin A+C (green signal in the nucleus), and laminin (red) in the TA muscle of CRISPR-targeted PDX DMD mice; nuclei were counterstained with DAPI (blue). Mice without editing (unedited) served as the negative control. Scale bar, 100 μm. b Representative box plots of the therapeutic efficacy of the different gene-editing strategies in vivo as determined by the ratio of the number of dystrophin-positive fibers (dystrophin + ) to that of lamin A+C-positive nuclei (LaminA+C + ); ∆46–54/Cas9, ∆46–54/Cas12a, and ∆45–55/Cas9 showed higher efficacy than INDEL50/Cas9 (P < 0.05, n = 8). c Confirmation of the presence of human cells (upper left) and reframing of mutant DMD in PDX DMD mice by PCR. ∆45–55 yielded an intron 44/intron 55 junction (upper right), and ∆46–54 yielded an intron 45/intron 54 junction (bottom row). hmtDNA, human mitochondrial DNA. d β-Dystroglycan restoration in human muscle fibers treated with different gene-editing strategies. Human dystrophin and β-dystroglycan are visible as green and red signals, respectively; sections were stained with DAPI (blue) to identify nuclei and labeled with an antibody against human Lamin A+C (green or red) to identify human nuclei. Mice without editing (unedited) served as a negative control. Scale bar, 50 μm

Article Snippet: CRISPR AAV vectors were generated by PackGene Biotech Co. (Guangzhou, China).

Techniques: In Vivo, Expressing, Immunofluorescence, CRISPR, Negative Control, Drug discovery, Mutagenesis, Staining, Labeling

Journal: Frontiers in Plant Science

Article Title: Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology

doi: 10.3389/fpls.2019.01446

Figure Lengend Snippet: The vector pBSE401 used for CRISPR/Cas9-mediated genome editing. AtU6, Arabidopsis U6 promotor; gRNA, guide RNA; 35S promotor, CaMV 35S promotor; Cas9, codon-optimized Cas9; NLS, nuclear location signal; bar , selective marker gene; KanR, Kanamycin resistance gene; pVS1-RepA, pVS1 replication origin; pVS1-StaA, pVS1 stability function.

Article Snippet: And then the CRISPR/Cas9 expression vector was transformed into E. coli Trans1 T1 (TransGen Biotech) used for soybean genetic transformation.

Techniques: Plasmid Preparation, CRISPR, Marker

Journal: Frontiers in Plant Science

Article Title: Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology

doi: 10.3389/fpls.2019.01446

Figure Lengend Snippet: Identifying transplants in T 0 generation. (A) Detection of the selectable marker gene bar by PAT/Bar test strip. The red arrowhead indicates that bar gene is positive. (B) Gel image of PCR products for T-DNA regions. Cas9, part of the Cas9 coding sequence. sgRNA, region from the U6 promoter to the downstream vector sequence spanning the sgRNA. GmActin was used as a normalization control. V: plasmid of the vector in transformation. WT, wild type soybean plants. Labels 1-16, individual mutant lines.

Article Snippet: And then the CRISPR/Cas9 expression vector was transformed into E. coli Trans1 T1 (TransGen Biotech) used for soybean genetic transformation.

Techniques: Marker, Stripping Membranes, Sequencing, Plasmid Preparation, Transformation Assay, Mutagenesis

Journal: Frontiers in Plant Science

Article Title: Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology

doi: 10.3389/fpls.2019.01446

Figure Lengend Snippet: CRISPR/Cas9-mediated targeted mutants of E1 in the T 1 generation.

Article Snippet: And then the CRISPR/Cas9 expression vector was transformed into E. coli Trans1 T1 (TransGen Biotech) used for soybean genetic transformation.

Techniques: CRISPR, Mutagenesis

Journal: Frontiers in Plant Science

Article Title: Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology

doi: 10.3389/fpls.2019.01446

Figure Lengend Snippet: CRISPR/Cas9-induced E1 mutants flowering time under both LD and SD conditions. (A) Phenotypes of wild type (WT, Jack) and homozygous T 2 mutant under LD condition, respectively. Top panel, WT did not have floral buds when T 2 mutant was flowering. Bottom panel, T 2 mutant produced the pods when WT was flowering. Red box, magnified view. (B) Flowering time of WT and homozygous T 2 mutants under LD condition. (C) Phenotypes of wild type (WT, Jack) and homozygous T 2 mutant under SD condition, respectively. (D) Flowering time of WT and homozygous T 2 mutants under SD condition. n, exact numbers of individual plants identified. **, homozygous T 2 mutants exhibit significant early flowering time (P < 0.01). The flowering time is shown as the mean values ± standard deviation.

Article Snippet: And then the CRISPR/Cas9 expression vector was transformed into E. coli Trans1 T1 (TransGen Biotech) used for soybean genetic transformation.

Techniques: CRISPR, Mutagenesis, Produced, Standard Deviation

Journal: Frontiers in Plant Science

Article Title: Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology

doi: 10.3389/fpls.2019.01446

Figure Lengend Snippet: Identifying of trans-clean mutants. (A) Detection of the selectable marker gene bar by PAT/Bar test strip. The red arrowhead indicates that bar gene is positive. (B) Gel image of PCR products for T-DNA elements. Cas9, part of the Cas9 coding sequence. sgRNA, region from the U6 promoter to the downstream vector sequence spanning the sgRNA. GmActin was used as a normalization control. V: plasmid of the vector in transformation. WT, wild type. Labels 1-20, individual mutant lines.

Article Snippet: And then the CRISPR/Cas9 expression vector was transformed into E. coli Trans1 T1 (TransGen Biotech) used for soybean genetic transformation.

Techniques: Marker, Stripping Membranes, Sequencing, Plasmid Preparation, Transformation Assay, Mutagenesis

Journal: BMC Biotechnology

Article Title: Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research

doi: 10.1186/s12896-018-0491-5

Figure Lengend Snippet: Sequencing analysis of CRISPR/Cas9-mediated TP53 knockout (KO) canine fatal fibroblasts. a Nucleotide sequences of targeted TP53 genomic loci of cells treated with TP53 gRNA #30 (colonies #2, #10, and #11) and b TP53 gRNA #39 (colonies #3, #5, and #6)

Article Snippet: CRISPR/Cas9 vectors targeting TP53 were transfected transiently using Polyexpress TM (Excellgene, Rockville, MD, USA) into K9 fetus 1 cells at passage 1 according to the manufacturer’s instructions.

Techniques: Sequencing, CRISPR, Knock-Out

Journal: BMC Biotechnology

Article Title: Establishment of TP53-knockout canine cells using optimized CRIPSR/Cas9 vector system for canine cancer research

doi: 10.1186/s12896-018-0491-5

Figure Lengend Snippet: Cellular characteristics of canine fibroblasts immortalized by CRISPR/Cas9-mediated TP53 knockout (KO). a No enhanced green fluorescent protein (EGFP) expression was detected by fluorescence activated cell sorting (FACS) analysis in both TP53KO#30 and TP53KO#39 cell lines. b Relative proliferation rates of each cell lines (× 15,000 cells). c Cumulative growth curve data of the control (early passage 2) and two TP53 KO cells were obtained after additional 14 consecutive passages in culture. d Representative images showing the cellular morphologies of the control and two TP53 KO cells at the end time-point (passage 14) of cumulative growth counting analysis. e Representative images of the control and two TP53 KO cells stained with senescence associated β-galactosidase (SA-β-gal) at the end time-point of cumulative growth counting analysis. f Percentage of SA-β-gal-positive cells in each group. g Immunoblot data showing TP53, p21, and SV40LT protein levels in the control, SV40LT-transduced, TP53KO#30, and TP53KO#39 cells grown in the absence or presence of 100 μM TMZ for 48 h. α-Tubulin was used as the loading control. **, p < 0.01

Article Snippet: CRISPR/Cas9 vectors targeting TP53 were transfected transiently using Polyexpress TM (Excellgene, Rockville, MD, USA) into K9 fetus 1 cells at passage 1 according to the manufacturer’s instructions.

Techniques: CRISPR, Knock-Out, Expressing, Fluorescence, FACS, Control, Staining, Western Blot