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1.
Cell ; 187(21): 5998-6015.e18, 2024 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-39191257

RESUMO

Internal states drive survival behaviors, but their neural implementation is poorly understood. Recently, we identified a line attractor in the ventromedial hypothalamus (VMH) that represents a state of aggressiveness. Line attractors can be implemented by recurrent connectivity or neuromodulatory signaling, but evidence for the latter is scant. Here, we demonstrate that neuropeptidergic signaling is necessary for line attractor dynamics in this system by using cell-type-specific CRISPR-Cas9-based gene editing combined with single-cell calcium imaging. Co-disruption of receptors for oxytocin and vasopressin in adult VMH Esr1+ neurons that control aggression diminished attack, reduced persistent neural activity, and eliminated line attractor dynamics while only slightly reducing overall neural activity and sex- or behavior-specific tuning. These data identify a requisite role for neuropeptidergic signaling in implementing a behaviorally relevant line attractor in mammals. Our approach should facilitate mechanistic studies in neuroscience that bridge different levels of biological function and abstraction.


Assuntos
Neurônios , Neuropeptídeos , Transdução de Sinais , Animais , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Camundongos , Masculino , Feminino , Neurônios/metabolismo , Sistemas CRISPR-Cas/genética , Ocitocina/metabolismo , Hipotálamo/metabolismo , Edição de Genes , Receptores de Vasopressinas/metabolismo , Receptores de Vasopressinas/genética , Camundongos Endogâmicos C57BL , Receptor alfa de Estrogênio/metabolismo
2.
Cell ; 187(3): 712-732.e38, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38194967

RESUMO

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.


Assuntos
Encéfalo , Organoides , Humanos , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Sistema Nervoso Central/metabolismo , Matriz Extracelular/metabolismo , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/citologia , Técnicas de Cultura de Tecidos , Células-Tronco/metabolismo , Morfogênese
3.
Cell ; 187(4): 861-881.e32, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38301646

RESUMO

Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy.


Assuntos
Antígeno B7-H1 , DNA Helicases , Imunidade Inata , Melanoma , Evasão Tumoral , Animais , Camundongos , Antígeno B7-H1/metabolismo , Instabilidade Genômica , Melanoma/imunologia , Melanoma/metabolismo , DNA Helicases/metabolismo
4.
Cell ; 186(18): 3983-4002.e26, 2023 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-37657419

RESUMO

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxΔRNaseH, the truncated editor used in dual-AAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed in vivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.


Assuntos
Bacteriófagos , Engenharia de Proteínas , Humanos , Animais , Camundongos , Bacteriófagos/genética , Encéfalo , Córtex Cerebral , RNA Polimerases Dirigidas por DNA
5.
Cell ; 186(2): 446-460.e19, 2023 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-36638795

RESUMO

Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knockins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens avenues in experimental T cell immunology.


Assuntos
Dependovirus , Engenharia Genética , Linfócitos T , Animais , Camundongos , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Marcação de Genes , Engenharia Genética/métodos
6.
Cell ; 186(21): 4567-4582.e20, 2023 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-37794590

RESUMO

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.


Assuntos
Sistemas CRISPR-Cas , Aberrações Cromossômicas , Edição de Genes , Linfócitos T , Humanos , Cromossomos , Sistemas CRISPR-Cas/genética , Dano ao DNA , Edição de Genes/métodos , Ensaios Clínicos como Assunto
7.
Cell ; 185(24): 4634-4653.e22, 2022 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-36347254

RESUMO

Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of ∼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes.


Assuntos
Sistemas CRISPR-Cas , Genes Essenciais , Humanos , Células HeLa , Técnicas de Inativação de Genes , Fenótipo
8.
Cell ; 185(6): 980-994.e15, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35303428

RESUMO

The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.


Assuntos
Toxinas Bacterianas , Clostridioides difficile , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Clostridioides difficile/genética , Lipoproteínas/genética
9.
Cell ; 184(22): 5622-5634.e25, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34610277

RESUMO

Disinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory, and excitatory neurons suggests that each circuit motif may be controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a genetically encoded GRP sensor, optogenetic anterograde stimulation, and trans-synaptic tracing, we reveal that GRP regulates VIP cells most likely via extrasynaptic diffusion from several local and long-range sources. In vivo photometry and CRISPR-Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.


Assuntos
Córtex Auditivo/metabolismo , Bombesina/metabolismo , Medo/fisiologia , Memória/fisiologia , Rede Nervosa/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Condicionamento Clássico , Peptídeo Liberador de Gastrina/química , Peptídeo Liberador de Gastrina/metabolismo , Regulação da Expressão Gênica , Genes Precoces , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Receptores da Bombesina/metabolismo , Som , Peptídeo Intestinal Vasoativo/metabolismo
10.
Cell ; 184(22): 5635-5652.e29, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34653350

RESUMO

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.


Assuntos
Edição de Genes , Sistemas CRISPR-Cas/genética , Linhagem Celular , DNA/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Feminino , Genes Dominantes , Genoma Humano , Humanos , Masculino , Modelos Biológicos , Proteína 1 Homóloga a MutL/genética , Mutação/genética , RNA/metabolismo , Reprodutibilidade dos Testes
11.
Cell ; 184(22): 5653-5669.e25, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34672952

RESUMO

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes. The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways. Systematic interrogation of this data uncovered unexpected relationships among DSB repair genes and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. This work provides a foundation for mapping DNA repair pathways and for optimizing genome editing across diverse modalities.


Assuntos
Quebras de DNA de Cadeia Dupla , Genômica , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular , Análise por Conglomerados , Reparo do DNA/genética , Edição de Genes , Regulação da Expressão Gênica , Genoma Humano , Humanos , Fenótipo , RNA Guia de Cinetoplastídeos/metabolismo , Reprodutibilidade dos Testes
12.
Cell ; 184(12): 3143-3162.e32, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34004147

RESUMO

Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.


Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Fosfatase 2/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Fosforilação , Ligação Proteica , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Especificidade por Substrato
13.
Cell ; 184(3): 709-722.e13, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33482084

RESUMO

Neural stem cells (NSCs) in the adult brain transit from the quiescent state to proliferation to produce new neurons. The mechanisms regulating this transition in freely behaving animals are, however, poorly understood. We customized in vivo imaging protocols to follow NSCs for several days up to months, observing their activation kinetics in freely behaving mice. Strikingly, NSC division is more frequent during daylight and is inhibited by darkness-induced melatonin signaling. The inhibition of melatonin receptors affected intracellular Ca2+ dynamics and promoted NSC activation. We further discovered a Ca2+ signature of quiescent versus activated NSCs and showed that several microenvironmental signals converge on intracellular Ca2+ pathways to regulate NSC quiescence and activation. In vivo NSC-specific optogenetic modulation of Ca2+ fluxes to mimic quiescent-state-like Ca2+ dynamics in freely behaving mice blocked NSC activation and maintained their quiescence, pointing to the regulatory mechanisms mediating NSC activation in freely behaving animals.


Assuntos
Células-Tronco Adultas/metabolismo , Cálcio/metabolismo , Ritmo Circadiano , Espaço Intracelular/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Comportamento Animal/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Citosol/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Melatonina/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Optogenética , Transdução de Sinais/efeitos dos fármacos , Triptaminas/farmacologia
14.
Cell ; 183(5): 1420-1435.e21, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33159857

RESUMO

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.


Assuntos
Bancos de Espécimes Biológicos , Tumores Neuroendócrinos/patologia , Organoides/patologia , Animais , Cromossomos Humanos/genética , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Masculino , Camundongos , Modelos Genéticos , Mutação/genética , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fenótipo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Transcriptoma/genética , Sequenciamento Completo do Genoma
15.
Cell ; 183(5): 1402-1419.e18, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33152263

RESUMO

We propose that the teratoma, a recognized standard for validating pluripotency in stem cells, could be a promising platform for studying human developmental processes. Performing single-cell RNA sequencing (RNA-seq) of 179,632 cells across 23 teratomas from 4 cell lines, we found that teratomas reproducibly contain approximately 20 cell types across all 3 germ layers, that inter-teratoma cell type heterogeneity is comparable with organoid systems, and teratoma gut and brain cell types correspond well to similar fetal cell types. Furthermore, cellular barcoding confirmed that injected stem cells robustly engraft and contribute to all lineages. Using pooled CRISPR-Cas9 knockout screens, we showed that teratomas can enable simultaneous assaying of the effects of genetic perturbations across all germ layers. Additionally, we demonstrated that teratomas can be sculpted molecularly via microRNA (miRNA)-regulated suicide gene expression to enrich for specific tissues. Taken together, teratomas are a promising platform for modeling multi-lineage development, pan-tissue functional genetic screening, and tissue engineering.


Assuntos
Linhagem da Célula , Modelos Biológicos , Teratoma/patologia , Animais , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , MicroRNAs/metabolismo , Reprodutibilidade dos Testes , Teratoma/genética
16.
Cell ; 180(6): 1262-1271.e15, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32169219

RESUMO

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.


Assuntos
Elementos Facilitadores Genéticos/genética , Ensaios de Triagem em Larga Escala/métodos , Polidactilia/genética , Animais , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Introdução de Genes/métodos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Mutação , Fenótipo , Polidactilia/metabolismo , RNA não Traduzido/genética
17.
Cell ; 182(2): 345-356.e16, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32589945

RESUMO

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.


Assuntos
Toxinas Bacterianas/metabolismo , Clostridium sordellii/metabolismo , Semaforinas/metabolismo , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Sítios de Ligação , Sistemas CRISPR-Cas/genética , Linhagem Celular , Microscopia Crioeletrônica , Edema/patologia , Edema/prevenção & controle , Feminino , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Semaforinas/química , Semaforinas/genética
18.
Annu Rev Biochem ; 88: 191-220, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30883196

RESUMO

Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Engenharia de Proteínas/métodos , RNA Guia de Cinetoplastídeos/genética , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Artefatos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Transferência de Genes , Genoma Humano , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Software
19.
Cell ; 176(1-2): 239-253.e16, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30503210

RESUMO

Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.


Assuntos
Proteínas Associadas a CRISPR/ultraestrutura , Sistemas CRISPR-Cas/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Proteínas de Bactérias , Proteínas Associadas a CRISPR/química , DNA/química , Desoxirribonucleases/metabolismo , Endorribonucleases/metabolismo , Modelos Moleculares , RNA/química , RNA Bacteriano/química , RNA Guia de Cinetoplastídeos/química , Ribonucleases/metabolismo
20.
Cell ; 179(1): 251-267.e24, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31539496

RESUMO

In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.


Assuntos
Neoplasias Encefálicas/genética , Modelos Animais de Doenças , Marcação de Genes/métodos , Loci Gênicos/genética , Glioma/genética , Mutagênese Insercional/métodos , Transgenes/genética , Animais , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco Neurais/metabolismo , Recombinases/metabolismo , Transfecção
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