Journal: The Journal of Cell Biology

Article Title: L1 retrotransposon antisense RNA within ASAR lncRNAs controls chromosome-wide replication timing

doi: 10.1083/jcb.201707082

Figure Lengend Snippet: Junctions and replication timing phenotypes of HTD114-derived cell clones with CRISPR/Cas9-mediated disruptions in ASAR6 L1PA2, chromosome 6: 96,206,263–96,212,288 (Feb 2009 [GRCh37/hg19])

Article Snippet: Using Lipofectamine 2000 according to the manufacturer’s recommendations, we cotransfected HTD114 cells with plasmids encoding GFP, sgRNAs, and Cas9 endonuclease (Origene).

Techniques: Clone Assay, CRISPR

Journal: PLoS Genetics

Article Title: Genetic compensation prevents myopathy and heart failure in an in vivo model of Bag3 deficiency

doi: 10.1371/journal.pgen.1009088

Figure Lengend Snippet: (A) Structure of the zebrafish bag3 gene and protein. Exon 2 is the target for the CRISPR/Cas9 gene editing in zebrafish bag3 . The CRISPR/Cas9-induced mutation (19 bp deletion) in bag3 is shown in bag3 mutant DNA sequencing chromatogram. The 19 nucleotides deletion in bag3 -/- leads to a frame shift, the introduction of a premature stop codon and thereby the premature termination of Bag3 translation, as demonstrated by the alignment of the Bag3 -/- and Bag3 +/+ aminoacid sequences (only partial aminoacid sequence shown) (B-C) Immunoblot analysis of 72 hpf bag3 +/+ embryo protein lysates compared to lysates obtained from bag3 -/- clutchmates with antibody against zebrafish Bag3. The figure shows one representative immunoblot from three independent experiments (N = 3, mean ± SD, P<0.0001 determined using two-tailed t-test). (D) Quantitative real-time PCR of bag3 +/+ and bag3 -/- embryos at 72 hpf shows significant downregulation of bag3 mRNA levels in bag3 -/- embryos (N = 3, mean ± SD, P = 0.0004 determined using two-tailed t-test).

Article Snippet: For CRISPR/Cas9 injections, 400 ng/μl recombinant Cas9 protein (Eupheria GmbH, Germany) was mixed with synthetic tracrRNA (100 ng/μl) and a gene-specific crRNA (Eurofins Genomics, Germany) against bag3 (50 ng/μl) in 200 mM KCl.

Techniques: CRISPR, Mutagenesis, DNA Sequencing, Sequencing, Western Blot, Two Tailed Test, Real-time Polymerase Chain Reaction

Journal: Biological Research

Article Title: Knockout of bcas3 gene causes neurodevelopment defects in zebrafish

doi: 10.1186/s40659-025-00615-4

Figure Lengend Snippet: Construction of bcas3 KO zebrafish by CRISPR/Cas9 technology. (A) , Whole-mount in situ hybridization (WISH) for bcas3 mRNA in WT zebrafish embryos at different development stages. Scale bars: 200 μm. ( n = 20 embryos per group). WT: wild type. (B) , Temporal pattern of mRNA expression of zebrafish bcas3 gene at different stages. mRNA extracts from whole-embryos were analyzed by RT-PCR for bcas3 mRNA levels at different zebrafish embryo stages. ( n = 20 embryos per group). (C) , Schematic diagram shows the genomic structure of zebrafish bcas3 with all 24 exons and the site of genome editing marked. The gene editing target site is located in the 12th exon of bcas3 gene. Target sequences are underlined. PAM sequences are highlighted in red. (D) , DNA sequence analysis identified the bcas3 −/− insertion mutant zebrafish line with a 22 bp insertion in exon 12 of bcas3 gene. (E) , The 22 bp insertion in bcas3 causes frame-shift and is predicted to cause a premature stop codon, resulting in a truncated mutant bcas3 proteins with only 302 amino acids left. (F-G) , Real-time RT-PCR analysis showed that the bcas3 mRNA level in 10 hpf (hours post fertilization) embryos and brains of 4 mpf (months post fertilization) bcas3 −/− zebrafish was reduced compared to WT controls. ( n = 3, per group). The rpl13a gene was used as endogenous control. Data are shown as mean ± SEM. Unpaired Student’s t-test was used to analyze RT-qPCR. Significance levels are denoted as follows: ** P < 0.01, *** P < 0.001

Article Snippet: Establishing zebrafish bcas3 -deficient mutant line was performed by microinjecting recombinant Cas9 protein (INNOBIO, Ningbo) and bcas3 -specific sgRNA.

Techniques: CRISPR, In Situ Hybridization, Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Mutagenesis, Quantitative RT-PCR, Control

Journal: Virology Journal

Article Title: Targeting the Kaposi’s sarcoma-associated herpesvirus genome with the CRISPR-Cas9 platform in latently infected cells

doi: 10.1186/s12985-021-01527-x

Figure Lengend Snippet: GFP expression in SLK-BAC16-mCherryORF45-infected cells following targeting of selected genes by CRISPR-Cas9. BAC16-mCherryORF45-infected SLK cells expressing Cas9 were transduced with recombinant lentiviruses encoding a random non-targeting sgRNA (control sgRNA) or sgRNAs targeting gfp , orf45 and orf73 . Each gene was targeted with a combination of two guides. Transduced cells were selected with 1 µg/ml puromycin and were grown in the absence or presence of 600 µg/ml hygromycin. Cells were harvested 6, 9, 13 and 16 days post transduction, and GFP expression was monitored by FACS analysis. Mock treated SLK-BAC16 cells that were either maintained in the presence or absence of hygromycin were used as controls. * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical test was performed between groups including all time points together

Article Snippet: These cells were transduced with a recombinant lentivirus encoding Cas9, selected with 7.5 μg/ml Blasticidin (A.G. Scientific Incorporation), and infected with a recombinant BAC16-based KSHV genome encoding N-terminal mCherry-tagged ORF45 which is expressed upon lytic induction [ ].

Techniques: Expressing, Infection, CRISPR, Transduction, Recombinant, Control

Journal: Virology Journal

Article Title: Targeting the Kaposi’s sarcoma-associated herpesvirus genome with the CRISPR-Cas9 platform in latently infected cells

doi: 10.1186/s12985-021-01527-x

Figure Lengend Snippet: Western blot analysis of GFP in SLK-BAC16-mCherry-ORF45-infected cells 16 days following targeting of selected genes by CRISPR-Cas9. BAC16-mCherry-ORF45-infected SLK cells expressing Cas9 were treated as described in Fig. . On day 16 post transduction, cells were collected, and protein extracts were prepared. Samples containing 40 µg of protein were used to determine GFP protein expression; anti-Tubulin was used as a loading control

Article Snippet: These cells were transduced with a recombinant lentivirus encoding Cas9, selected with 7.5 μg/ml Blasticidin (A.G. Scientific Incorporation), and infected with a recombinant BAC16-based KSHV genome encoding N-terminal mCherry-tagged ORF45 which is expressed upon lytic induction [ ].

Techniques: Western Blot, Infection, CRISPR, Expressing, Transduction, Control

Journal: Virology Journal

Article Title: Targeting the Kaposi’s sarcoma-associated herpesvirus genome with the CRISPR-Cas9 platform in latently infected cells

doi: 10.1186/s12985-021-01527-x

Figure Lengend Snippet: Viral DNA quantification following CRISPR-Cas9 targeting of gfp , orf45 or orf73 . BAC16-mCherry-ORF45-infected SLK cells expressing Cas9 were treated as described in Fig. . DNA was extracted and subjected to TaqMan real-time PCR for viral DNA using primers that target the viral orfK6 gene along with the cellular erv-1 gene, which was used to normalize loading. Statistical test was performed between groups including all time points together for each gRNA treatment. * p < 0.05, *** p < 0.001

Article Snippet: These cells were transduced with a recombinant lentivirus encoding Cas9, selected with 7.5 μg/ml Blasticidin (A.G. Scientific Incorporation), and infected with a recombinant BAC16-based KSHV genome encoding N-terminal mCherry-tagged ORF45 which is expressed upon lytic induction [ ].

Techniques: CRISPR, Infection, Expressing, Real-time Polymerase Chain Reaction

Journal: Virology Journal

Article Title: Targeting the Kaposi’s sarcoma-associated herpesvirus genome with the CRISPR-Cas9 platform in latently infected cells

doi: 10.1186/s12985-021-01527-x

Figure Lengend Snippet: Visualization of LANA dots and Western blot analysis of LANA 16 days following targeting of orf73 by CRISPR-Cas9. Cas9-BAC16-mCherryORF45-infected SLK cells were transduced with recombinant lentiviruses expressing a random non-targeting sgRNA or orf73 sgRNAs. Cells were maintained with no hygromycin selection, fixed 16 days post transduction and stained with rat monoclonal anti-LANA followed by anti-rat Alexa Fluor 647-conjugated secondary antibodies. The corresponding staining of nuclear DNA by Hoechst is also displayed ( a ). BAC16-mCherryORF45-infected SLK cells expressing Cas9 were treated as described in Fig. . Cells were collected 16 days post transduction, and protein extracts were prepared. Samples containing 60 µg of protein extracts from cells that were maintained with hygromycin and with no hygromycin selection, respectively, were used to determine LANA protein expression while Tubulin was used as loading control ( b )

Article Snippet: These cells were transduced with a recombinant lentivirus encoding Cas9, selected with 7.5 μg/ml Blasticidin (A.G. Scientific Incorporation), and infected with a recombinant BAC16-based KSHV genome encoding N-terminal mCherry-tagged ORF45 which is expressed upon lytic induction [ ].

Techniques: Western Blot, CRISPR, Infection, Transduction, Recombinant, Expressing, Selection, Staining, Control

Journal: Virology Journal

Article Title: Targeting the Kaposi’s sarcoma-associated herpesvirus genome with the CRISPR-Cas9 platform in latently infected cells

doi: 10.1186/s12985-021-01527-x

Figure Lengend Snippet: Sequencing the episome CRISPR-Cas9 cut sites. To map insertions and deletions (indels), we amplified genomic DNA and subjected it to deep sequencing. The percentage on the y axis represents the total number of reads with indels divided by the total numbers of reads. The identities of the samples are indicated on the x axis, including the name of the gene and the number of days post sgRNA transduction. Percentage of indels flanking the cutting site in gfp using the hygromycin minus sgGFP samples ( a ). Percentage of indels flanking the cutting site in orf73 using both hygromycin minus and hygromycin plus samples ( b ). Percentage of indels around the cutting site in orf45 using hygromycin minus samples ( c )

Article Snippet: These cells were transduced with a recombinant lentivirus encoding Cas9, selected with 7.5 μg/ml Blasticidin (A.G. Scientific Incorporation), and infected with a recombinant BAC16-based KSHV genome encoding N-terminal mCherry-tagged ORF45 which is expressed upon lytic induction [ ].

Techniques: Sequencing, CRISPR, Amplification, Transduction

Journal: Virology Journal

Article Title: Targeting the Kaposi’s sarcoma-associated herpesvirus genome with the CRISPR-Cas9 platform in latently infected cells

doi: 10.1186/s12985-021-01527-x

Figure Lengend Snippet: Distribution and abundance of deletions flanking CRISPR-Cas9 cut sites. The graphs show the percentage of nucleotide deletion based on amplicon deep sequencing. Cutting site in orf73 ( a – h ), cutting site in gfp hygromycin minus ( i – l ) and no guide was used ( a , b , i , j ) (control). ( a , c , e , g , i , k ) the graphs show the most abundant deletion length. ( b , d , f , h , j, l ) the graph shows the distribution of deletions around the cutting site (0). Time points are indicated in the title. a , c , e , g , i , k the x-axis represent the length of the most highly frequent deletions. b , d , f , h , j , l the x-axis represent the frequency of a base-pair to be deleted, in both sides of the cutting cite (0- between nucleotides 3 and 4 5′ to the pam sequence

Article Snippet: These cells were transduced with a recombinant lentivirus encoding Cas9, selected with 7.5 μg/ml Blasticidin (A.G. Scientific Incorporation), and infected with a recombinant BAC16-based KSHV genome encoding N-terminal mCherry-tagged ORF45 which is expressed upon lytic induction [ ].

Techniques: CRISPR, Amplification, Sequencing, Control