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

Journal: Cancer Discovery

Article Title: Transcriptional Silencing of ALDH2 Confers a Dependency on Fanconi Anemia Proteins in Acute Myeloid Leukemia

doi: 10.1158/2159-8290.cd-20-1542

Figure Lengend Snippet: Figure 3. Inactivation of FA genes in AML leads to p53-induced cell-cycle arrest and apoptosis. A, Representative flow cytometry analysis of BrdU incorporation and DNA content to infer cell status following lentiviral transduction of MOLM-13 cells with the indicated sgRNAs (day 6). B, Quantifica- tion of different cell-cycle stages, average of three biological replicates. Paired Student t test was applied to calculate P values. C, Representative flow cytometry analysis of DAPI (indicating permeable dead cells) and Annexin-V staining (a preapoptotic cell marker) following lentiviral transduction of MOLM-13 cells (day 6). D, Quantification of live and apoptotic cells, average of three biological replicates. Paired Student t test was applied to calculate P values. E, Gene set enrichment analysis of RNA-seq data obtained from MOLM-13 cells lentivirally transduced with the indicated sgRNAs (70). Normalized enrichment score (NES) and family-wise error rate (FWER) P value are shown. F, Western blot analysis performed on lysates obtained from MOLM-13 cells on day 6 following sgRNA transduction. G, Competition-based proliferation assays in MOLM-13 cells following sequential sgRNA transduc- tion. Negative sgRNA or TP53 sgRNAs were infected first, selected with neomycin, followed by transduction with the sgRNAs indicated at the bottom of the graph (linked with GFP). n = 3. All bar graphs represent the mean ± SEM. All sgRNA experiments were performed in Cas9-expressing cell lines.

Article Snippet: Plasmid Construction: sgRNA and shRNA Cloning For CRISPR screening, the optimized sgRNA lentiviral expression vector (LRG2.1T) and the lentiviral human codon–optimized Streptococcus pyogenes Cas9 vector (LentiV_Cas9_Puro, Addgene: 108100) were used.

Techniques: Flow Cytometry, BrdU Incorporation Assay, Transduction, Staining, Marker, RNA Sequencing, Western Blot, Infection, Expressing

Journal: Nature Communications

Article Title: PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice

doi: 10.1038/s41467-019-09180-3

Figure Lengend Snippet: A PiggyBac transposon system for recessive screening in mice. a Structure of "inactivating transposons" ITP1 and ITP2 . b Transgenic mouse lines generated using ITP1 or ITP2 . Transposon copy numbers within the transgene array as well as the chromosomal donor locus of the array are shown. Chromosomal donor loci for different lines are shown on the right, as detected by fluorescence in situ hybridization with transposon-specific probes. Note that the ITP2-M and ITP2-N mouse lines emerged from a single founder animal. c Structures of the Rosa26 PB and Blm m3 alleles as described earlier , . The Rosa26 PB knock-in allele expresses the insect version of the PiggyBac transposase constitutively driven by the endogenous Rosa26 promoter. d , e Kaplan–Meier plots showing survival of IPB and control mice. In d the whole cohort is shown ( n = 123 [IPB], n = 87 [controls]; p < 0.0001, log-rank test). e represents survival curves for mice displaying hematopoietic tumors characterized histopathologically ( n = 59 [IPB], n = 16 [controls]; p = 0.025, log-rank test). PB PiggyBac, SB Sleeping Beauty, Av-SA adenovirus-derived splice acceptor, bGEO β-galactosidase/neomycin resistance reporter including the bovine growth hormone polyadenylation signal, En2-SA Engrailed-2 exon-2 splice acceptor, pA SV40 bidirectional polyadenylation signal, Tp transposon, R26 Rosa26, SA splice acceptor, Blm Bloom syndrome RecQ like helicase, nd not done

Article Snippet: To generate Rosa26 Cas9 knock-in mice (Gt(ROSA)26Sor tm1(Cas9)Rrad ) , we cloned a human codon-optimized hemagglutinin-tagged Cas9 sequence derived from Streptococcus pyogenes into a Gateway-compatible entry vector (Addgene #17398 ), which was then shuttled into a Rosa26 -targeting Gateway destination vector with loxP-flanked puromycin resistance-containing stop cassette (modified after Addgene #21189 ).

Techniques: Transgenic Assay, Generated, Fluorescence, In Situ Hybridization, Knock-In, Control, Derivative Assay

Journal: Nature Communications

Article Title: PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice

doi: 10.1038/s41467-019-09180-3

Figure Lengend Snippet: Characterization and classification of DLBCLs from IPB mice. a Immunohistochemical characterization and sub-classification of mouse DLBCLs ( n = 25) from ITP2-M;Rosa26 PB/+ ;Blm m3/m3 (IPB) mice. Microscopic images of two representative DLBCL cases, one classified as germinal center B-cell like (GCB) DLBCL (left panel) and the other as non-GCB DLBCL (right panel). Tumors consist of large-sized neoplastic cells, show strong expression of B220/CD45R (B-cell marker), and are negative for CD138 (plasma cell marker). While the GCB DLBCL sample exhibits high expression of Bcl6 and is negative for Mum1/Irf4, the non-GCB DLBCL case shows the opposite expression pattern. Both tumors show high proliferation rates as indicated by Ki-67 immunohistochemistry (IHC). Scale bar, 50 µm. b RNA sequencing-based sub-classification of DLBCLs ( n = 25) from IPB mice. For clustering of the expression data, mouse orthologues to human DLBCL classifier genes were used. The heatmap shows z-transformed expression values. Two main clusters (A and B) were identified. IHC-based subtyping (GCB/non-GCB) of the corresponding tumors is indicated at the top of the heatmap. GEP gene expression profiling, ABC activated B-cell like

Article Snippet: To generate Rosa26 Cas9 knock-in mice (Gt(ROSA)26Sor tm1(Cas9)Rrad ) , we cloned a human codon-optimized hemagglutinin-tagged Cas9 sequence derived from Streptococcus pyogenes into a Gateway-compatible entry vector (Addgene #17398 ), which was then shuttled into a Rosa26 -targeting Gateway destination vector with loxP-flanked puromycin resistance-containing stop cassette (modified after Addgene #21189 ).

Techniques: Immunohistochemical staining, Expressing, Marker, Clinical Proteomics, Immunohistochemistry, RNA Sequencing, Transformation Assay, Gene Expression

Journal: Nature Communications

Article Title: PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice

doi: 10.1038/s41467-019-09180-3

Figure Lengend Snippet: Clonality analysis of DLBCLs by immune repertoire sequencing. a Workflow for B-cell receptor repertoire analysis. Full-length amplification and sequencing of immunoglobulin heavy and light chain variable regions was performed from bulk tumor tissue ( n = 30) of ITP2-M;Rosa26 PB/+ ;Blm m3/m3 (IPB) mice. Top image shows exemplary cDNA product after library preparation with amplified variable and constant region of the immunoglobulin heavy chain (light gray). Unique molecular identifiers (UMI; green) and adapters and barcodes for sequencing (dark gray) were introduced during library preparation. Dotted arrows indicate reads for 300 bp paired-end sequencing. Sequenced raw reads were de-multiplexed and a consensus read sequence for each UMI was assembled with MIGEC . All reads containing an identical UMI were collapsed into one read. MiXCR was used for mapping of reads to mouse reference sequences and clonotype assembly based on the complementarity-determining region 3 (CDR3) region. To visualize the clonal structure of individual tumors, we developed CloNet , a pipeline for generation of clonality network plots. b Exemplary clonality network plots derived from four different mouse DLBCL samples. Plots display clonal structures of immunoglobulin heavy and light chains. Each clone (defined by a unique CDR3 sequence) constitutes a node of the clonality network. The size of the node scales with the third root of the count of the reads assigned to it. A link between two nodes was drawn if the clones mapped to identical V and J genes and differed by at most 1 bp in their CDR3 sequence. The complexity of the branching of a clone (i.e. number of subclones) is a measure for the grade of somatic hypermutation. Clones defined by a unique V(D)J rearrangement that contained more than 10% of the total reads are highlighted in color. Two monoclonal ( IPB_10.6c and IPB_11.4c ) samples, one biclonal ( IPB_10.3c ) and one oligoclonal ( IPB_1.5c ) sample are shown. c Proportion of monoclonal (1 clone), biclonal (2 clones), and oligo-/polyclonal (>2 clones) DLBCL samples. BC barcode, V variable gene segment, NDN diversity gene segment, J joining gene segment, C constant gene segment, HC heavy chain, LC light chain, Tr total reads, Cf fraction of clone

Article Snippet: To generate Rosa26 Cas9 knock-in mice (Gt(ROSA)26Sor tm1(Cas9)Rrad ) , we cloned a human codon-optimized hemagglutinin-tagged Cas9 sequence derived from Streptococcus pyogenes into a Gateway-compatible entry vector (Addgene #17398 ), which was then shuttled into a Rosa26 -targeting Gateway destination vector with loxP-flanked puromycin resistance-containing stop cassette (modified after Addgene #21189 ).

Techniques: Sequencing, Amplification, Derivative Assay, Clone Assay

Journal: Nature Communications

Article Title: PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice

doi: 10.1038/s41467-019-09180-3

Figure Lengend Snippet: Genetic analysis of DLBCLs. a Scheme of experimental and analytic workflow. b Overlay of copy number profiles showing cancer-relevant amplifications/deletions in 16 DLBCLs from ITP2-M;Rosa26 PB/+ ; Blm m3/m3 (IPB) mice. A zoomed-in view is provided for Chr11, which harbors frequent amplifications (identified in mouse GCB as well as ABC DLBCLs). The minimal amplified region contains—among other genes—the known DLBCL oncogenes Bcl11a and Rel . c Circos plot visualizing transposon insertion data from 42 IPB-DLBCLs. Rings from inward to outward: Insertion density plot for both orientations (shown in blue and green), number of insertions per common insertion site (CIS; dark yellow bars; axis from 0 to 140), number of contributing samples per CIS (red bars; axis from 0 to 40). Top 50 CIS genes are annotated. d Top 50 CIS genes ranked by number of contributing DLBCL samples. Molecular function (determined by literature search) of CIS genes is indicated. Transcription factors constituted the largest functional gene class ( n = 15). Compared to the total number of mouse transcription factors ( n = 1603), the enrichment for transcription factors among the top 50 CIS genes is highly significant ( p = 1.4 10 −5 , Fisher’s exact test). Genes present in Cancer Gene Census (CGC) database and/or known for their role in DLBCL (literature search) are represented in dark blue, unknown DLBCL genes by light blue boxes. QiSeq quantitative transposon insertion site sequencing, aCGH array comparative genomic hybridization

Article Snippet: To generate Rosa26 Cas9 knock-in mice (Gt(ROSA)26Sor tm1(Cas9)Rrad ) , we cloned a human codon-optimized hemagglutinin-tagged Cas9 sequence derived from Streptococcus pyogenes into a Gateway-compatible entry vector (Addgene #17398 ), which was then shuttled into a Rosa26 -targeting Gateway destination vector with loxP-flanked puromycin resistance-containing stop cassette (modified after Addgene #21189 ).

Techniques: Amplification, Functional Assay, Sequencing, Hybridization

Journal: Nature Communications

Article Title: PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice

doi: 10.1038/s41467-019-09180-3

Figure Lengend Snippet: Loss of heterozygosity analysis in tumors from IPB mice. a Loss of heterozygosity (LOH) analysis workflow. Fragment of Pten containing the single nucleotide polymorphism (SNP) rs30424206 was amplified and sequenced in tumors and tails from ITP2-M;Rosa26 PB/+ ; Blm m3/m3 (IPB) mice ( n = 10). b Variant frequencies determined by Pten SNP sequencing of tail and tumor tissue samples from six IPB mice. All tumors harbored high-coverage Pten insertions. c Pten IHC of tumors from IPB mice ( n = 10), as well as from DLBCLs generated in a Blm -proficient screen using ATP2-S1;Rosa26 PB/+ mice ( n = 7). Pten expression was scored semi-quantitatively. Representative microscopic images are shown. Pie charts display proportions of tumor samples with different Pten expression scores. Scale bar, 50 µm; Insets, ×200 magnification

Article Snippet: To generate Rosa26 Cas9 knock-in mice (Gt(ROSA)26Sor tm1(Cas9)Rrad ) , we cloned a human codon-optimized hemagglutinin-tagged Cas9 sequence derived from Streptococcus pyogenes into a Gateway-compatible entry vector (Addgene #17398 ), which was then shuttled into a Rosa26 -targeting Gateway destination vector with loxP-flanked puromycin resistance-containing stop cassette (modified after Addgene #21189 ).

Techniques: Amplification, Variant Assay, Sequencing, Generated, Expressing

Journal: Nature Communications

Article Title: PiggyBac transposon tools for recessive screening identify B-cell lymphoma drivers in mice

doi: 10.1038/s41467-019-09180-3

Figure Lengend Snippet: A CRISPR/Cas9-based in vivo platform for BCL functional genomics. a Outline of the functional genomic approach for in vivo gene validation. b Kaplan–Meier plot showing survival of mice transplanted with hematopoietic stem/progenitor cells (HSPC) transduced with Trp53 sgRNA, Trp53 shRNA, and non-targeting (NT) sgRNA. p < 0.0001 for both Trp53-sgRNA vs. NT-sgRNA and Trp53-shRNA vs. NT-sgRNA. p < 0.03 for Trp53-sgRNA vs. Trp53-shRNA. p -values (log-rank test) were corrected for multiple testing using the Bonferroni method. c The transposon insertion pattern in Rfx7 predicts that gene-disruption is the cancer-causing mechanism. Each arrow represents an individual insertion. Insertions from all DLBCLs in the cohort are shown. ITP2 transposons can trap genes in either orientation. Insertions are distributed over the whole length of the gene (predicting a tumor suppressor). There is no bias for hot-spot areas of insertions as typically observed for unidirectional "activating" insertions in oncogenes. Consensus coding sequence ( Rfx7-201 ) is displayed. d Kaplan–Meier plot for mice transplanted with HSPCs transduced with Rfx7 sgRNA and NT sgRNA. p < 0.0001, log-rank test. e Transposon insertion pattern in Phip , predicting tumor suppressive function of the gene. Consensus coding sequence ( Phip-201 ) is shown. Each arrow represents an individual insertion. Insertions from all DLBCLs in the cohort are shown. f Heatmap displaying copy number variations on human chromosome 6 in samples from the TCGA-DLBC ( n = 48) dataset (TCGA Research Network: http://cancergenome.nih.gov). The position of PHIP is indicated. g Function of Phip as a B-cell lymphoma tumor suppressor was validated using the CRISPR/Cas9-based in vivo functional genomic approach. Kaplan–Meier plot for mice transplanted with HSPCs transduced with Phip sgRNA and non-targeting (NT) sgRNA transplants. p < 0.0001, log-rank test. sgRNA single guide RNA, EFS elongation factor 1-alpha core promoter, eGFP enhanced green fluorescent protein, Mb megabase

Article Snippet: To generate Rosa26 Cas9 knock-in mice (Gt(ROSA)26Sor tm1(Cas9)Rrad ) , we cloned a human codon-optimized hemagglutinin-tagged Cas9 sequence derived from Streptococcus pyogenes into a Gateway-compatible entry vector (Addgene #17398 ), which was then shuttled into a Rosa26 -targeting Gateway destination vector with loxP-flanked puromycin resistance-containing stop cassette (modified after Addgene #21189 ).

Techniques: CRISPR, In Vivo, Functional Assay, Biomarker Discovery, Transduction, shRNA, Disruption, Sequencing