Journal: Nucleic Acids Research

Article Title: Translocations can drive expression changes of multiple genes in regulons covering entire chromosome arms

doi: 10.1093/nar/gkaf677

Figure Lengend Snippet: Gene expression analysis in cells carrying translocation t(11;14). ( A ) Fraction of upregulated protein-coding genes in MCL patients versus mature healthy B cells per chromosome. The dashed line represents the expected fraction. ( B ) Volcano plot of protein-coding genes in chr11 down. The dashed lines show the absolute log2 FC threshold of 1, and the –log10 value of FDR 0.1. ( C ) As in B, but for chr11 up. ( D ) Location of up- and downregulated genes in MCL patients on chr11 down. Names for upregulated genes are depicted. Below, compartment scores of each Mb bin in MCL and healthy B cells are shown. ( E ) As D, but for chr11 up. ( F ) Scheme of in vitro translocation generation. ( G ) Interaction frequencies from different viewpoints to chr11 down measured by 4C-seq. Vertical bars indicate the breakpoint proximal (chr11: 69 500 000 – 73 500 000), YAP1 (chr11: 100 000 000 – 104 000 000), and CD3 (chr11: 116 500 000 – 120 500 000) region. ( H ) PCA of samples harboring translocations generated in vitro, and ( I ) volcano plot of gene expression changes, both based on bulk RNA-seq. ( J ) UMAP of samples harboring translocations generated in vitro, based on scRNA-seq. ( K ) Fraction of upregulated protein-coding genes per chromosome in CCND1-positive versus -negative cells, after in vitro induction of translocations. The dashed line represents the expected fraction. wt = wild-type, down = downstream of the breakpoint, up = upstream of the breakpoint, MCL = mantle cell lymphoma, HBC = healthy B cells.

Article Snippet: Single guide RNA molecules were designed to target the region where translocations usually cluster in MCL patients and synthesized by Synthego (Redwood City, CA, USA).

Techniques: Gene Expression, Translocation Assay, In Vitro, Generated, RNA Sequencing

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