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