المؤلفون: Tan, I-Li, Perez, Alexendar, Lew, Rachel, Sun, Xiaoyu, Baldwin, Alisha, Zhu, Yong, Shah, Mihir, Berger, Mitchel, Doudna, Jennifer, Fellmann, Christof

المصدر: Cell Reports. 42(11)

مصطلحات موضوعية: CP: Cancer, CRISPR-Cas9, cancer shredding, genome shredding, glioblastoma, hypermutated glioma, Humans, Temozolomide, Brain Neoplasms, Cell Line, Tumor, Drug Resistance, Neoplasm, Neoplasm Recurrence, Local, Glioblastoma, Glioma, Antineoplastic Agents, Alkylating

الوصف: Glioblastoma (GBM) is the most common lethal primary brain cancer in adults. Despite treatment regimens including surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy, growth of residual tumor leads to therapy resistance and death. At recurrence, a quarter to a third of all gliomas have hypermutated genomes, with mutational burdens orders of magnitude greater than in normal tissue. Here, we quantified the mutational landscape progression in a patients primary and recurrent GBM, and we uncovered Cas9-targetable repeat elements. We show that CRISPR-mediated targeting of highly repetitive loci enables rapid elimination of GBM cells, an approach we term genome shredding. Importantly, in the patients recurrent GBM, we identified unique repeat sequences with TMZ mutational signature and demonstrated that their CRISPR targeting enables cancer-specific cell ablation. Cancer shredding leverages the non-coding genome and therapy-induced mutational signatures for targeted GBM cell depletion and provides an innovative paradigm to develop treatments for hypermutated glioma.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/20n4j5s8Test

المؤلفون: Hamilton, Jennifer R, Tsuchida, Connor A, Nguyen, David N, Shy, Brian R, McGarrigle, E Riley, Sandoval Espinoza, Cindy R, Carr, Daniel, Blaeschke, Franziska, Marson, Alexander, Doudna, Jennifer A

المصدر: Cell Reports. 35(9)

مصطلحات موضوعية: Biological Sciences, Biotechnology, Genetics, Transplantation, Gene Therapy, 5.2 Cellular and gene therapies, Development of treatments and therapeutic interventions, Quality Education, A549 Cells, CD4-Positive T-Lymphocytes, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Cell Engineering, Gene Editing, Gene Transfer Techniques, HIV-1, Humans, Jurkat Cells, Lentivirus, Receptors, Chimeric Antigen, Ribonucleoproteins, Transgenes, Virion, env Gene Products, Human Immunodeficiency Virus, CAR-T cells, CRISPR delivery, CRISPR-Cas9, precision genome editing, viral engineering, virus-like particles, Biochemistry and Cell Biology, Medical Physiology, Biological sciences

الوصف: As genome engineering advances cell-based therapies, a versatile approach to introducing both CRISPR-Cas9 ribonucleoproteins (RNPs) and therapeutic transgenes into specific cells would be transformative. Autologous T cells expressing a chimeric antigen receptor (CAR) manufactured by viral transduction are approved to treat multiple blood cancers, but additional genetic modifications to alter cell programs will likely be required to treat solid tumors and for allogeneic cellular therapies. We have developed a one-step strategy using engineered lentiviral particles to introduce Cas9 RNPs and a CAR transgene into primary human T cells without electroporation. Furthermore, programming particle tropism allows us to target a specific cell type within a mixed cell population. As a proof-of-concept, we show that HIV-1 envelope targeted particles to edit CD4+ cells while sparing co-cultured CD8+ cells. This adaptable approach to immune cell engineering ex vivo provides a strategy applicable to the genetic modification of targeted somatic cells in vivo.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/3k21j58nTest

المؤلفون: Tambe, Akshay, East-Seletsky, Alexandra, Knott, Gavin J, Doudna, Jennifer A, O’Connell, Mitchell R

المصدر: Cell Reports. 24(4)

مصطلحات موضوعية: Biochemistry and Cell Biology, Biological Sciences, Genetics, CRISPR-Associated Proteins, CRISPR-Cas Systems, Carrier Proteins, Endonucleases, Humans, RNA, Guide, Kinetoplastida, C2c2, CRISPR-Cas systems, Cas13, Cas13a, RNA biology, RNA specificity, Medical Physiology, Biological sciences

الوصف: CRISPR-Cas13a enzymes are RNA-guided, RNA-activated RNases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging, and RNA regulation. However, the relationship between target RNA binding and HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activation is poorly understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number and the position of mismatches between the guide and the target. We identify a central binding seed for which perfect base pairing is required for target binding and a separate nuclease switch for which imperfect base pairing results in tight binding, but not HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13a RNA binding and cleavage behavior for RNA-targeting tool development.

الوصول الحر: https://escholarship.org/uc/item/1mv316hnTest

المؤلفون: Blair, John D, Hockemeyer, Dirk, Doudna, Jennifer A, Bateup, Helen S, Floor, Stephen N

المصدر: Cell Reports. 21(7)

مصطلحات موضوعية: Biological Sciences, Bioinformatics and Computational Biology, Genetics, Neurosciences, Stem Cell Research - Nonembryonic - Human, Human Genome, Stem Cell Research, Regenerative Medicine, Stem Cell Research - Embryonic - Human, Underpinning research, 1.1 Normal biological development and functioning, Neurological, 3' Untranslated Regions, Cell Line, Cells, Cultured, Gene Expression Regulation, Developmental, Humans, Neural Stem Cells, Neurogenesis, Prosencephalon, Protein Biosynthesis, Proteome, TOR Serine-Threonine Kinases, Transcriptome, RNA, TrIP-seq, cellular differentiation, human stem cell, neural progenitor cell, neurogenesis, neuron, polysome profiling, ribosome profiling, translational control, 3’ Untranslated Regions, Biochemistry and Cell Biology, Medical Physiology, Biological sciences

الوصف: Faithful cellular differentiation requires temporally precise activation of gene expression programs, which are coordinated at the transcriptional and translational levels. Neurons express the most complex set of mRNAs of any human tissue, but translational changes during neuronal differentiation remain incompletely understood. Here, we induced forebrain neuronal differentiation of human embryonic stem cells (hESCs) and measured genome-wide RNA and translation levels with transcript-isoform resolution. We found that thousands of genes change translation status during differentiation without a corresponding change in RNA level. Specifically, we identified mTOR signaling as a key driver for elevated translation of translation-related genes in hESCs. In contrast, translational repression in active neurons is mediated by regulatory sequences in 3' UTRs. Together, our findings identify extensive translational control changes during human neuronal differentiation and a crucial role of 3' UTRs in driving cell-type-specific translation.

الوصول الحر: https://escholarship.org/uc/item/8rq2098sTest

المؤلفون: Hultquist, Judd F, Schumann, Kathrin, Woo, Jonathan M, Manganaro, Lara, McGregor, Michael J, Doudna, Jennifer, Simon, Viviana, Krogan, Nevan J, Marson, Alexander

المصدر: Cell Reports. 17(5)

مصطلحات موضوعية: Biological Sciences, Biotechnology, Genetics, Sexually Transmitted Infections, HIV/AIDS, Infectious Diseases, 2.1 Biological and endogenous factors, Aetiology, Infection, CD4-Positive T-Lymphocytes, CRISPR-Cas Systems, Cells, Cultured, Gene Editing, Gene Knockout Techniques, HIV Infections, HIV-1, Host-Pathogen Interactions, Humans, Intercellular Signaling Peptides and Proteins, Receptors, CXCR4, Receptors, CXCR5, Reproducibility of Results, Ribonucleoproteins, beta Karyopherins, CCR5, CRISPR/Cas9, CXCR4, HIV integrase, LEDGF, TNPO3, genome editing, host dependency factors, host-pathogen interactions, primary T cells, Biochemistry and Cell Biology, Medical Physiology, Biological sciences

الوصف: New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4+ T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here, we adapted this methodology to a high-throughput platform for the efficient, arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner, whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously, enabling studies of interactions among multiple host and viral factors. Finally, in an arrayed screen of 45 genes associated with HIV integrase, we identified several candidate dependency/restriction factors, demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/4j48n7zmTest
https://escholarship.org/content/qt4j48n7zm/qt4j48n7zm.pdfTest

المؤلفون: Kranzusch, Philip J, Lee, Amy Si-Ying, Berger, James M, Doudna, Jennifer A

المصدر: Cell Reports. 3(5)

مصطلحات موضوعية: Biochemistry and Cell Biology, Biological Sciences, Genetics, Prevention, Emerging Infectious Diseases, Vaccine Related, Biodefense, Underpinning research, 1.1 Normal biological development and functioning, Inflammatory and immune system, 2', 5'-Oligoadenylate Synthetase, Amino Acid Sequence, Biocatalysis, Catalytic Domain, Crystallography, X-Ray, DNA, HEK293 Cells, Humans, Immunity, Innate, Interferons, Membrane Proteins, Molecular Sequence Data, Nucleotidyltransferases, Protein Binding, Recombinant Proteins, Second Messenger Systems, Medical Physiology, Biological sciences

الوصف: Innate immune recognition of foreign nucleic acids induces protective interferon responses. Detection of cytosolic DNA triggers downstream immune signaling through activation of cyclic GMP-AMP synthase (cGAS). We report here the crystal structure of human cGAS, revealing an unanticipated zinc-ribbon DNA-binding domain appended to a core enzymatic nucleotidyltransferase scaffold. The catalytic core of cGAS is structurally homologous to the RNA-sensing enzyme, 2'-5' oligo-adenylate synthase (OAS), and divergent C-terminal domains account for specific ligand-activation requirements of each enzyme. We show that the cGAS zinc ribbon is essential for STING-dependent induction of the interferon response and that conserved amino acids displayed within the intervening loops are required for efficient cytosolic DNA recognition. These results demonstrate that cGAS and OAS define a family of innate immunity sensors and that structural divergence from a core nucleotidyltransferase enables second-messenger responses to distinct foreign nucleic acids.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/1c74m9cqTest