Journal: Poultry Science

Article Title: Multiple pathways to evaluate the immunoprotective effect of Turkeys Herpesvirus recombinant vaccine expressing HA of H9N2

doi: 10.1016/j.psj.2024.104335

Figure Lengend Snippet: Construction of HVT recombinant virus expressing HA gene of H9N2. (A) HVT-EGFP-HA recombinant viruses were constructed through both the NHEJ and HDR pathways, respectively. Cas9: CRISPR-associated protein 9, Sg-A: A 20-bp sequence from a prokaryotic organism, crRNA: CRISPR-derived RNA, tracrRNA: trans-activating crRNA, HA: Hemagglutinin, EGFP: enhanced Green Fluorescent Protein, US: short unique, NHEJ: nonhomologous end joining, HDR: homology-directed recombination, UL: long unique, TRL: terminal UL repeats, IRL: internal UL repeats, IRS: internal US repeats, TRS: terminal US repeats. (B) The EGFP expression cassette was knocked out using Cre recombinase to obtain HVT-HA recombinant virus. cre: cyclization recombination enzyme. (C) Comparison of the number of HVT-EGFP-HA recombinant viruses constructed by NHEJ and HDR.

Article Snippet: The DNA oligos of gRNA were annealed and synthesized and cloned into the CRISPR/Cas9 vector pX459 (Addgene, Cambridge, MA) using BbsI cloning site.

Techniques: Recombinant, Virus, Expressing, Construct, CRISPR, Sequencing, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: The Mechanism of MICU-Dependent Gating of the Mitochondrial Ca 2+ Uniporter

doi: 10.1101/2020.04.04.025833

Figure Lengend Snippet: ( A ) A schematic arrangement of various subunits in the MCU complex. Four MCU and four EMRE subunits form the pore of the MCU complex (only two MCU and two EMRE subunits are shown for simplicity). EMRE also tethers MICU1 subunit to the pore on the cytosolic side of the IMM (i.e., in the mitochondrial intermembrane space, IMS). MICU1 forms homodimers or hetero-dimerizes with MICU2 or MICU3 (not shown). Each MICU subunit has two EF hands that bind cytosolic Ca 2+ . ( B to F ) CRISPR-mediated indels in various MCU subunit genes and the resulting mutant alleles. The CRISPR binding sites (for sgRNA) are highlighted in yellow , and their PAM sequences are highlighted in green . The translational initiation codon (ATG) is shown in bold where applicable. (B) Overview of the MCU gene and indels in the knockout. A sgRNA was used to target exon 3. The sequence of targeted region in MCU gene is shown; exon 3 is underlined. Targeted sequencing indicates frame-shift indels ( red ) in both alleles ( Al- 1 and Al- 2). (C) Overview of the EMRE gene and truncated region in the knockout. Two sgRNAs were used for CRISPR-Cas9–mediated deletion in the exon-2 ( underlined ) and the flanking region. Targeted sequencing indicates same 259-bp deletion ( red ) in both alleles. (D) Overview of the MICU1 gene and truncated region in the knockout. Two sgRNAs were used for CRISPR-Cas9– mediated deletion in the exon-3 ( underlined ) and the flanking region. Targeted sequencing indicates that almost all of exon-3 is deleted along with a portion of the flanking region ( red ) in both alleles ( Al- 1 and A l- 2). (E) Overview of the MICU2 gene and truncated region in the knockout. Two sgRNAs were used for CRISPR-Cas9–mediated deletion in the exon-1 ( underlined ) and the flanking region. Targeted sequencing indicates that almost all of exon-1 is deleted ( red ) in both alleles. (F) Overview of the MICU3 gene and truncated region in the knockout. Two sgRNAs were used for CRISPR-Cas9–mediated deletion in the exon-1 ( underlined ). Targeted sequencing indicates a 73-bp deletion in the expected cut area ( red ) in both alleles.

Article Snippet: MEFs were transfected with the Cas9 gRNA vector (Addgene: PX459) via electroporation (Invitrogen Neon transfection system) using the following parameters: 1×10 6 cells and 1 μg of two different gRNA-Cas9 plasmids.

Techniques: CRISPR, Mutagenesis, Binding Assay, Knock-Out, Sequencing