Journal: Advanced Science

Article Title: KMT2D Deficiency Promotes Myeloid Leukemias which Is Vulnerable to Ribosome Biogenesis Inhibition

doi: 10.1002/advs.202206098

Figure Lengend Snippet: Kmt2d mutation by CRISPR/Cas9 promotes AML in mice. A) Schematic experimental design for mouse modeling using CRISPR/Cas9 system. Trp53 −/− ; Cas9 mouse HSPCs were transduced with mCherry‐linked sg Kmt2d ‐sg Nf1 ‐sg Cas9 or sgScramble‐sg Nf1 ‐sg Cas9 , and then transplanted into sub‐lethally irradiated syngeneic mice. B) Kaplan–Meier survival curves of mice transplanted with Trp53 −/− ; Cas9 HSPCs transduced with sgScramble‐sg Nf1 ‐sg Cas9 (blue; n = 5), sg Kmt2d_ #1‐sg Nf1 ‐sg Cas9 (red, n = 5), or sg Kmt2d_# 2‐sg Nf1 ‐sg Cas9 (orange; n = 5). ** p < 0.01 (log‐rank test). C) WBC, Hb, and PLT counts of sgScramble and sg Kmt2d mice 2 months post‐transplant. Graph represents the mean ± SD; ** p < 0.01, *** p < 0.001, ns, not significant (unpaired two‐tailed t ‐test). D) Representative flow cytometric profiles showing the expression of CD11b/Gr‐1, B220/CD3ε and c‐Kit in bone marrow cells of sacrificed TNKC ( Trp53 −/− ; sg Nf1 ; sg Kmt2d; sg Cas9 ) mice. E) Representative images of histological analyses of blood, spleen, liver, and bone marrow of sacrificed TNKC mice. F) T7 endonuclease I assay on Kmt2d in bone marrow cells of sacrificed TNKC mice. G) Mutation analyses of the Kmt2d regions targeted by CRISPR/Cas9 of TNKC bone marrow cells. Representative Sanger sequences of single TA clones.

Article Snippet: sgRNAs were designed by the ATUM CRISPR gRNA Design tool ( https://www.atum.bio/eCommerce/cas9/input ).

Techniques: Mutagenesis, CRISPR, Transduction, Irradiation, Two Tailed Test, Expressing, T7EI Assay, Clone Assay

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