Your browser does not allow saving data (e.g. private mode): bookmarks will be lost when the tab closes.
Search PubMed
Keywords, author, journal, years — results from PubMed, abstracts with Russian translation, bookmarks in your browser.
Found: 26 125
Page 215 of 1000-
Structures of the holo CRISPR RNA-guided transposon integration complex.
CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism1-3. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.
-
Genomic sequencing and editing revealed the GRM8 signaling pathway as potential therapeutic targets of squamous cell lung cancer.
The study sought to explore novel genetic aberration driving squamous cell lung carcinoma (LUSC). The whole exome (WES), whole genome (WGS) and target region (TS) sequencings and CRISPR-Cas9 genome editing techniques were integrated to explore and validate novel targeting candidates from LUSC primary tumors and corresponding patient-derived xenografts (PDXs). Seven genes (FGFR2, GRM1,PIK3CG, PIK3CA,ZFHX4, CSMD3, GRM8) with high frequencies of both single nucleotide variants (SNVs) and copy number variants (CNVs), and two genes (CLDN1 and RIT1) only with CNVs were identified by bioinformatics analysis. The functions of these candidates were validated through CRISPR-Cas9 system in primary PDX cells. Furthermore, we focused on the genetic and functional analysis of Metabotropic glutamate receptor 8 (GRM8), whose transcriptional activation was elucidated to promote the survival of LUSC tumor cell through inhibiting cAMP pathway and activating MAPK pathway. The SNV identified in GRM8, A112G, activated downstream signaling pathway and induced cell proliferation, which could be reversed by cAMP stimulator and MEK inhibitor. In conclusion, the components of GRM8 signaling pathway could serve as potential targets of squamous cell lung cancer carrying GRM8 activating variants.
-
Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays.
CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration.
-
Live-cell imaging reveals the dynamics of PRC2 and recruitment to chromatin by SUZ12-associated subunits.
Polycomb-repressive complex 2 (PRC2) is a histone methyltransferase that promotes epigenetic gene silencing, but the dynamics of its interactions with chromatin are largely unknown. Here we quantitatively measured the binding of PRC2 to chromatin in human cancer cells. Genome editing of a HaloTag into the endogenous EZH2 and SUZ12 loci and single-particle tracking revealed that ∼80% of PRC2 rapidly diffuses through the nucleus, while ∼20% is chromatin-bound. Short-term treatment with a small molecule inhibitor of the EED-H3K27me3 interaction had no immediate effect on the chromatin residence time of PRC2. In contrast, separation-of-function mutants of SUZ12, which still form the core PRC2 complex but cannot bind accessory proteins, revealed a major contribution of AEBP2 and PCL homolog proteins to chromatin binding. We therefore quantified the dynamics of this chromatin-modifying complex in living cells and separated the contributions of H3K27me3 histone marks and various PRC2 subunits to recruitment of PRC2 to chromatin.
-
AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice.
CRISPR editing of muscle stem cells (MuSCs) with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies. However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9.Cre into Ai14 mice. These mice express tdTomato upon Cre-mediated removal of a floxed stop codon. About 28%-47% and 24%-89% of Pax7+ MuSCs expressed tdTomato in pre-grafts and regenerated grafts (p > 0.05), respectively, suggesting AAV9 efficiently transduced MuSCs, and AAV9-edited MuSCs renewed successfully. Robust MuSC transduction was further confirmed by delivering AAV9.Cre to Pax7-ZsGreen-Ai14 mice in which Pax7+ MuSCs are genetically labeled by ZsGreen. Next, we co-injected AAV9.Cas9 and AAV9.gRNA to dystrophic mdx mice to repair the mutated dystrophin gene. CRISPR-treated and untreated muscles were grafted to immune-deficient, dystrophin-null NSG.mdx4cv mice. Grafts regenerated from CRISPR-treated muscle contained the edited genome and yielded 2.7-fold more dystrophin+ cells (p = 0.015). Importantly, increased dystrophin expression was not due to enhanced formation of revertant fibers or de novo transduction by residual CRISPR vectors in the graft. We conclude that AAV9 effectively transduces MuSCs. AAV9 CRISPR editing of MuSCs may provide enduring therapy.
-
TREML4 receptor regulates inflammation and innate immune cell death during polymicrobial sepsis.
Sepsis is a biphasic disease characterized by an acute inflammatory response, followed by a prolonged immunosuppressive phase. Therapies aimed at controlling inflammation help to reduce the time patients with sepsis spend in intensive care units, but they do not lead to a reduction in overall mortality. Recently, the focus has been on addressing the immunosuppressive phase, often caused by apoptosis of immune cells. However, molecular triggers of these events are not yet known. Using whole-genome CRISPR screening in mice, we identified a triggering receptor expressed on myeloid cells (TREM) family receptor, TREML4, as a key regulator of inflammation and immune cell death in sepsis. Genetic ablation of Treml4 in mice demonstrated that TREML4 regulates calcium homeostasis, the inflammatory cytokine response, myeloperoxidase activation, the endoplasmic reticulum stress response and apoptotic cell death in innate immune cells, leading to an overall increase in survival rate, both during the acute and chronic phases of polymicrobial sepsis.
-
Mutations in Plasmodium falciparum actin-binding protein coronin confer reduced artemisinin susceptibility.
Drug resistance is an obstacle to global malaria control, as evidenced by the recent emergence and rapid spread of delayed artemisinin (ART) clearance by mutant forms of the PfKelch13 protein in Southeast Asia. Identifying genetic determinants of ART resistance in African-derived parasites is important for surveillance and for understanding the mechanism of resistance. In this study, we carried out long-term in vitro selection of two recently isolated West African parasites (from Pikine and Thiès, Senegal) with increasing concentrations of dihydroartemisinin (DHA), the biologically active form of ART, over a 4-y period. We isolated two parasite clones, one from each original isolate, that exhibited enhanced survival to DHA in the ring-stage survival assay. Whole-genome sequence analysis identified 10 mutations in seven different genes. We chose to focus on the gene encoding PfCoronin, a member of the WD40-propeller domain protein family, because mutations in this gene occurred in both independent selections, and the protein shares the β-propeller motif with PfKelch13 protein. For functional validation, when pfcoronin mutations were introduced into the parental parasites by CRISPR/Cas9-mediated gene editing, these mutations were sufficient to reduce ART susceptibility in the parental lines. The discovery of a second gene for ART resistance may yield insights into the molecular mechanisms of resistance. It also suggests that pfcoronin mutants could emerge as a nonkelch13 type of resistance to ART in natural settings.
-
Rapid interrogation of cancer cell of origin through CRISPR editing.
The increasing complexity of different cell types revealed by single-cell analysis of tissues presents challenges in efficiently elucidating their functions. Here we show, using prostate as a model tissue, that primary organoids and freshly isolated epithelial cells can be CRISPR edited ex vivo using Cas9-sgRNA (guide RNA) ribotnucleoprotein complex technology, then orthotopically transferred in vivo into immunocompetent or immunodeficient mice to generate cancer models with phenotypes resembling those seen in traditional genetically engineered mouse models. Large intrachromosomal (∼2 Mb) or multigenic deletions can be engineered efficiently without the need for selection, including in isolated subpopulations to address cell-of-origin questions.
-
Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana.
Precise genetic modification can be achieved via a sequence homology-mediated process known as gene targeting (GT). Whilst established for genome engineering purposes, the application of GT in plants still suffers from a low efficiency for which an explanation is currently lacking. Recently reported reduced rates of GT in A. thaliana deficient in polymerase theta (Polθ), a core component of theta-mediated end joining (TMEJ) of DNA breaks, have led to the suggestion of a direct involvement of this enzyme in the homology-directed process. Here, by monitoring homology-driven gene conversion in plants with CRISPR reagent and donor sequences pre-integrated at random sites in the genome (in planta GT), we demonstrate that Polθ action is not required for GT, but instead suppresses the process, likely by promoting the repair of the DNA break by end-joining. This finding indicates that lack of donor integration explains the previously established reduced GT rates seen upon transformation of Polθ-deficient plants. Our study additionally provides insight into ectopic gene targeting (EGT), recombination events between donor and target that do not map to the target locus. EGT, which occurs at similar frequencies as "true" GT during transformation, was rare in our in planta GT experiments arguing that EGT predominantly results from target locus recombination with nonintegrated T-DNA molecules. By describing mechanistic features of GT our study provides directions for the improvement of precise genetic modification of plants.
-
The L348P point mutation in cardiac myosin binding protein-C alters transient responses to stretch, slows cardiac relaxation, and is embryonic lethal in homozygous CRISPR gene-edited mice.
Mutations in cardiac myosin binding protein-C (cMyBP-C) are a common cause of hypertrophic cardiomyopathy (HCM), an inherited autosomal dominant disease affecting 1 in 250-500 people. We previously identified a single amino acid substitution (L348P) in the regulatory motif (M-domain) of cMyBP-C that slowed relaxation and caused diastolic dysfunction in transgenic mice. Here we attempted to increase expression of the mutant protein by creating a CRISPR gene-edited knock-in mouse model (L348P-CR) and breeding mice to homozygosity for the mutant allele. Results showed that L348P-CR homozygous mice died in utero, but that heterozygous knock-in mice developed contractile deficits and diastolic dysfunction comparable to transgenic mice. To overcome the lethal homozygous expression of the L348P mutation, we used our "cut-and-paste" approach to fully replace endogenous wild-type cMyBP-C with recombinant L348P cMyBP-C in permeabilized cardiomyocytes from SpyC3 mice. Results showed that replacement of wild-type cMyBP-C with recombinant L348P recapitulated mechanical effects seen in transgenic and L348P-CR mice, validating the utility of our cut-and-paste method for evaluating functional effects of cMyBP-C. We conclude that L348P-CR knock-in mice are a robust model of diastolic dysfunction due to a single point mutation in cMyBP-C and that the cut-and-paste approach offers a rapid and cost-effective approach for evaluating mutations in cMyBP-C, especially those that are lethal in traditional animal models.
PubMed returns only the first 10,000 results — refine the query to see the rest.