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1.
Cell ; 185(22): 4067-4081.e21, 2022 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-36306733

RESUMO

The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.


Assuntos
Sistemas CRISPR-Cas , RNA Guia de Cinetoplastídeos , RNA Guia de Cinetoplastídeos/metabolismo , Endonucleases/metabolismo , Pareamento de Bases , Nucleotídeos , Edição de Genes
2.
Mol Cell ; 81(17): 3637-3649.e5, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34478654

RESUMO

The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Linfócitos T/metabolismo , Proteína 9 Associada à CRISPR/fisiologia , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/fisiologia , DNA/genética , Endonucleases/genética , Edição de Genes/métodos , Técnicas Genéticas , Genoma/genética , Genômica/métodos , Humanos , Leucócitos Mononucleares/metabolismo , Conformação Molecular , RNA Guia de Cinetoplastídeos/genética , Relação Estrutura-Atividade , Linfócitos T/fisiologia
3.
Cytotherapy ; 25(7): 750-762, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37086241

RESUMO

BACKGROUND AIMS: Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. METHODS: To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. RESULTS: These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. CONCLUSIONS: Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos de Linfócitos T , Receptor de Morte Celular Programada 1/metabolismo , Linhagem Celular Tumoral , Linfócitos T , Imunoterapia Adotiva
4.
Mol Cell ; 56(2): 333-339, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25373540

RESUMO

The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner and has been widely used for programmable genome editing. Cas9 activity is dependent on interactions with guide RNAs, and evolutionarily divergent Cas9 nucleases have been shown to work orthogonally. However, the molecular basis of selective Cas9:guide-RNA interactions is poorly understood. Here, we identify and characterize six conserved modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity. We show the bulge and nexus are necessary for DNA cleavage and demonstrate that the nexus and hairpins are instrumental in defining orthogonality between systems. In contrast, the crRNA:tracrRNA complementary region can be modified or partially removed. Collectively, our results establish guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.


Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Clivagem do DNA , DNA/química , Endonucleases/química , Engenharia Genética/métodos , RNA Guia de Cinetoplastídeos/química , Proteína 9 Associada à CRISPR , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Células HEK293 , Humanos , Conformação de Ácido Nucleico , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/ultraestrutura
6.
Nat Methods ; 14(6): 600-606, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28459459

RESUMO

RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.


Assuntos
Sistemas CRISPR-Cas/genética , Mapeamento Cromossômico/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA
7.
Nature ; 505(7482): 239-43, 2014 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-24291791

RESUMO

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-L-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l(-1)) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.


Assuntos
Biocombustíveis/provisão & distribuição , Metabolismo dos Carboidratos , Etanol/metabolismo , Engenharia Genética , Phaeophyceae/metabolismo , Saccharomyces cerevisiae/metabolismo , Alginatos/metabolismo , Anaerobiose , Ascomicetos/genética , Ascomicetos/metabolismo , Biotecnologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Evolução Molecular , Fermentação , Teste de Complementação Genética , Ácido Glucurônico/metabolismo , Ácidos Hexurônicos/metabolismo , Manitol/metabolismo , Phaeophyceae/genética , Ácido Quínico/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Alga Marinha/genética , Alga Marinha/metabolismo , Ácidos Urônicos/metabolismo
8.
Cancer Immunol Res ; 12(4): 462-477, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38345397

RESUMO

Allogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here, we describe preclinical data supporting the ongoing first-in-human clinical study, the CaMMouflage trial (NCT05722418), evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic, allogeneic anti-B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition, T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell-mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M-HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst, p. 385.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/terapia , Antígeno de Maturação de Linfócitos B/metabolismo , Antígenos HLA-E , Linfócitos T , Receptores de Antígenos de Linfócitos T , Imunoterapia Adotiva , Antígenos de Histocompatibilidade Classe I/metabolismo , Aloenxertos/patologia
9.
Arch Biochem Biophys ; 489(1-2): 110-7, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19664586

RESUMO

The Thiobacillus denitrificans genome contains two sequences corresponding to ATP sulfurylase (Tbd_0210 and Tbd_0874). Both genes were cloned and expressed protein characterized. The larger protein (Tbd_0210; 544 residues) possesses an N-terminal ATP sulfurylase domain and a C-terminal APS kinase domain and was therefore annotated as a bifunctional enzyme. But, the protein was not bifunctional because it lacked ATP sulfurylase activity. However, the enzyme did possess APS kinase activity and displayed substrate inhibition by APS. Truncated protein missing the N-terminal domain had <2% APS kinase activity suggesting the function of the inactive sulfurylase domain is to promote the oligomerization of the APS kinase domains. The smaller gene product (Tbd_0874; 402 residues) possessed strong ATP sulfurylase activity with kinetic properties that appear to be kinetically optimized for the direction of APS utilization and ATP+sulfate production, which is consistent with an enzyme that functions physiologically to produce inorganic sulfate.


Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/química , Sulfato Adenililtransferase/química , Thiobacillus/enzimologia , Clonagem Molecular , Expressão Gênica , Cinética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Sulfato Adenililtransferase/genética , Thiobacillus/genética
10.
Nat Biotechnol ; 37(12): 1471-1477, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740839

RESUMO

Type I CRISPR-Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5-7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8-11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI-Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade-Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR-Cas systems can be harnessed for genome engineering applications in eukaryotic cells.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Escherichia coli , Genoma/genética , Células HEK293 , Humanos , Modelos Genéticos
11.
Trends Biotechnol ; 36(2): 134-146, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28778606

RESUMO

The term 'clustered regularly interspaced short palindromic repeats' (CRISPR) has recently become synonymous with the genome-editing revolution. The RNA-guided endonuclease CRISPR-associated protein 9 (Cas9), in particular, has attracted attention for its promise in basic research and gene editing-based therapeutics. CRISPR-Cas systems are efficient and easily programmable nucleic acid-targeting tools, with uses reaching beyond research and therapeutic development into the precision breeding of plants and animals and the engineering of industrial microbes. CRISPR-Cas systems have potential for many microbial engineering applications, including bacterial strain typing, immunization of cultures, autoimmunity or self-targeted cell killing, and the engineering or control of metabolic pathways for improved biochemical synthesis. In this review, we explore the fundamental characteristics of CRISPR-Cas systems and highlight how these features can be used in industrial settings.


Assuntos
Biotecnologia/métodos , Sistemas CRISPR-Cas , Edição de Genes/métodos , Microbiologia Industrial/métodos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR , Endonucleases/genética , Endonucleases/metabolismo , Engenharia Genética/métodos , Redes e Vias Metabólicas/genética , Plantas/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
12.
Genome Biol ; 19(1): 214, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518407

RESUMO

BACKGROUND: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery. RESULTS: Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5'-N4GATT-3'), for NmeCas9 genome editing in human cells. CONCLUSIONS: Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Edição de Genes/métodos , Neisseria meningitidis/enzimologia , Animais , Humanos
13.
Biochemistry ; 47(6): 1608-21, 2008 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-18205391

RESUMO

The essential enzymatic cofactor NAD+ can be synthesized in many eukaryotes, including Saccharomyces cerevisiae and mammals, using tryptophan as a starting material. Metabolites along the pathway or on branches have important biological functions. For example, kynurenic acid can act as an NMDA antagonist, thereby functioning as a neuroprotectant in a wide range of pathological states. N-Formyl kynurenine formamidase (FKF) catalyzes the second step of the NAD+ biosynthetic pathway by hydrolyzing N-formyl kynurenine to produce kynurenine and formate. The S. cerevisiae FKF had been reported to be a pyridoxal phosphate-dependent enzyme encoded by BNA3. We used combined crystallographic, bioinformatic and biochemical methods to demonstrate that Bna3p is not an FKF but rather is most likely the yeast kynurenine aminotransferase, which converts kynurenine to kynurenic acid. Additionally, we identify YDR428C, a yeast ORF coding for an alpha/beta hydrolase with no previously assigned function, as the FKF. We predicted its function based on our interpretation of prior structural genomics results and on its sequence homology to known FKFs. Biochemical, bioinformatics, genetic and in vivo metabolite data derived from LC-MS demonstrate that YDR428C, which we have designated BNA7, is the yeast FKF.


Assuntos
Arilformamidase/metabolismo , Saccharomyces cerevisiae/enzimologia , Transaminases/metabolismo , Sequência de Aminoácidos , Arilformamidase/química , Cromatografia Líquida de Alta Pressão , Biologia Computacional , Cristalografia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Transaminases/química
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