RESUMO
Genome editing has been a transformative force in the life sciences and human medicine, offering unprecedented opportunities to dissect complex biological processes and treat the underlying causes of many genetic diseases. CRISPR-based technologies, with their remarkable efficiency and easy programmability, stand at the forefront of this revolution. In this Review, we discuss the current state of CRISPR gene editing technologies in both research and therapy, highlighting limitations that constrain them and the technological innovations that have been developed in recent years to address them. Additionally, we examine and summarize the current landscape of gene editing applications in the context of human health and therapeutics. Finally, we outline potential future developments that could shape gene editing technologies and their applications in the coming years.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Disciplinas das Ciências Biológicas , Terapia Genética , TecnologiaRESUMO
Protein structures are essential to understanding cellular processes in molecular detail. While advances in artificial intelligence revealed the tertiary structure of proteins at scale, their quaternary structure remains mostly unknown. We devise a scalable strategy based on AlphaFold2 to predict homo-oligomeric assemblies across four proteomes spanning the tree of life. Our results suggest that approximately 45% of an archaeal proteome and a bacterial proteome and 20% of two eukaryotic proteomes form homomers. Our predictions accurately capture protein homo-oligomerization, recapitulate megadalton complexes, and unveil hundreds of homo-oligomer types, including three confirmed experimentally by structure determination. Integrating these datasets with omics information suggests that a majority of known protein complexes are symmetric. Finally, these datasets provide a structural context for interpreting disease mutations and reveal coiled-coil regions as major enablers of quaternary structure evolution in human. Our strategy is applicable to any organism and provides a comprehensive view of homo-oligomerization in proteomes.
Assuntos
Inteligência Artificial , Proteínas , Proteoma , Humanos , Proteínas/química , Proteínas/genética , Archaea/química , Archaea/genética , Eucariotos/química , Eucariotos/genética , Bactérias/química , Bactérias/genéticaRESUMO
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 GenesRESUMO
De novo design of complex protein folds using solely computational means remains a substantial challenge1. Here we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from G-protein-coupled receptors2, are not found in the soluble proteome, and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses demonstrate the high thermal stability of the designs, and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, as a proof of concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we have designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.
Assuntos
Desenho Assistido por Computador , Aprendizado Profundo , Proteínas de Membrana , Dobramento de Proteína , Solubilidade , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Estabilidade Proteica , Proteoma/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Estudo de Prova de ConceitoRESUMO
Physical interactions between proteins are essential for most biological processes governing life1. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic and structural data increase. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications2-9. Here we use a geometric deep-learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features that are critical to drive protein-protein interactions10. We hypothesized that these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof of principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1 and CTLA-4. Several designs were experimentally optimized, whereas others were generated purely in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling an approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.
Assuntos
Simulação por Computador , Aprendizado Profundo , Ligação Proteica , Proteínas , Humanos , Proteínas/química , Proteínas/metabolismo , Proteômica , Mapas de Interação de Proteínas , Sítios de Ligação , Biologia SintéticaRESUMO
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/fisiologiaRESUMO
Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage1-3. The programmable activity of Cas9 has been widely utilized for genome editing applications4-6, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , Domínios Proteicos , Estruturas R-Loop , Streptococcus pyogenes , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/ultraestrutura , Catálise , DNA/metabolismo , Clivagem do DNA , Ativação Enzimática , Edição de Genes , RNA Guia de Cinetoplastídeos/metabolismo , Streptococcus pyogenes/enzimologia , Especificidade por SubstratoRESUMO
Macrophages are a diverse group of phagocytic cells acting in host protection against stress, injury, and pathogens. Here, we show that the scavenger receptor SR-A6 is an entry receptor for human adenoviruses in murine alveolar macrophage-like MPI cells, and important for production of type I interferon. Scavenger receptors contribute to the clearance of endogenous proteins, lipoproteins and pathogens. Knockout of SR-A6 in MPI cells, anti-SR-A6 antibody or the soluble extracellular SR-A6 domain reduced adenovirus type-C5 (HAdV-C5) binding and transduction. Expression of murine SR-A6, and to a lower extent human SR-A6 boosted virion binding to human cells and transduction. Virion clustering by soluble SR-A6 and proximity localization with SR-A6 on MPI cells suggested direct adenovirus interaction with SR-A6. Deletion of the negatively charged hypervariable region 1 (HVR1) of hexon reduced HAdV-C5 binding and transduction, implying that the viral ligand for SR-A6 is hexon. SR-A6 facilitated macrophage entry of HAdV-B35 and HAdV-D26, two important vectors for transduction of hematopoietic cells and human vaccination. The study highlights the importance of scavenger receptors in innate immunity against human viruses.
Assuntos
Infecções por Adenoviridae/virologia , Pulmão/virologia , Macrófagos Alveolares/virologia , Macrófagos/virologia , Receptores Imunológicos/metabolismo , Receptores Imunológicos/fisiologia , Internalização do Vírus , Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/metabolismo , Adenovírus Humanos/imunologia , Animais , Humanos , Imunidade Inata , Pulmão/imunologia , Pulmão/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Receptores Imunológicos/genéticaRESUMO
Replication across damaged DNA templates is accompanied by transient formation of sister chromatid junctions (SCJs). Cells lacking Esc2, an adaptor protein containing no known enzymatic domains, are defective in the metabolism of these SCJs. However, how Esc2 is involved in the metabolism of SCJs remains elusive. Here we show interaction between Esc2 and a structure-specific endonuclease Mus81-Mms4 (the Mus81 complex), their involvement in the metabolism of SCJs, and the effects Esc2 has on the enzymatic activity of the Mus81 complex. We found that Esc2 specifically interacts with the Mus81 complex via its SUMO-like domains, stimulates enzymatic activity of the Mus81 complex in vitro, and is involved in the Mus81 complex-dependent resolution of SCJs in vivo Collectively, our data point to the possibility that the involvement of Esc2 in the metabolism of SCJs is, in part, via modulation of the activity of the Mus81 complex.
Assuntos
Cromátides/metabolismo , DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular , Cromátides/química , Clonagem Molecular , Dano ao DNA , Replicação do DNA , DNA Cruciforme/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Instabilidade Genômica , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismoRESUMO
The sliding clamp, PCNA, plays a central role in DNA replication and repair. In the moving replication fork, PCNA is present at the leading strand and at each of the Okazaki fragments that are formed on the lagging strand. PCNA enhances the processivity of the replicative polymerases and provides a landing platform for other proteins and enzymes. The loading of the clamp onto DNA is performed by the Replication Factor C (RFC) complex, whereas its unloading can be carried out by an RFC-like complex containing Elg1. Mutations in ELG1 lead to DNA damage sensitivity and genome instability. To characterize the role of Elg1 in maintaining genomic integrity, we used homology modeling to generate a number of site-specific mutations in ELG1 that exhibit different PCNA unloading capabilities. We show that the sensitivity to DNA damaging agents and hyper-recombination of these alleles correlate with their ability to unload PCNA from the chromatin. Our results indicate that retention of modified and unmodified PCNA on the chromatin causes genomic instability. We also show, using purified proteins, that the Elg1 complex inhibits DNA synthesis by unloading SUMOylated PCNA from the DNA. Additionally, we find that mutations in ELG1 suppress the sensitivity of rad5Δ mutants to DNA damage by allowing trans-lesion synthesis to take place. Taken together, the data indicate that the Elg1-RLC complex plays an important role in the maintenance of genomic stability by unloading PCNA from the chromatin.
Assuntos
Proteínas de Transporte/genética , Dano ao DNA , Instabilidade Genômica , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , DNA/biossíntese , DNA Helicases/genética , Metanossulfonato de Metila/toxicidade , Mutação , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/química , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Supressão GenéticaRESUMO
Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Reparo de DNA por Recombinação , Motivos de Aminoácidos , DNA/biossíntese , DNA Polimerase III/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Células HEK293 , Humanos , Ubiquitina-Proteína Ligases/fisiologia , Raios UltravioletaRESUMO
BACKGROUND: Adenoviruses are common pathogens infecting animals and humans. They are classified based on serology, or genome sequence information. These methods have limitations due to lengthy procedures or lack of infectivity data. Adenoviruses are easy to produce and amenable to genetic and biochemical modifications, which makes them a powerful tool for biological studies, and clinical gene-delivery and vaccine applications. Antibodies directed against adenoviral proteins are important diagnostic tools for virus identification in vivo and in vitro, and are used to elucidate infection mechanisms, often in combination with genomic sequencing and type specific information from hyper-variable regions of structural proteins. RESULTS: Here we describe a novel and readily useable method for cloning, expressing and purifying small fragments of hyper-variable regions 1-6 of the adenoviral hexon protein. We used these polypeptides as antigens for generating polyclonal rabbit antibodies against human adenovirus 3 (HAdV-B3), mouse adenovirus 1 (MAdV-1) and MAdV-2 hexon. In Western immunoblots with lysates from cells infected from thirteen human and three mouse viruses, these antibodies bound to homologous full-length hexon protein and revealed variable levels of cross-reactivity to heterologous hexons. Results from immuno-fluorescence and electron microscopy studies indicated that HAdV-B3 and MAdV-2 hexon antibodies recognized native forms of hexon. CONCLUSIONS: The procedure described here can in principle be applied to any adenovirus for which genome sequence information is available. It provides a basis for generating novel type-specific tools in diagnostics and research, and extends beyond the commonly used anti-viral antibodies raised against purified viruses or subviral components.
Assuntos
Adenoviridae/genética , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas Recombinantes/imunologia , Células A549 , Infecções por Adenoviridae/diagnóstico , Infecções por Adenoviridae/imunologia , Adenovírus Humanos/genética , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/isolamento & purificação , Sequência de Bases , Western Blotting , Capsídeo/imunologia , Capsídeo/ultraestrutura , Proteínas do Capsídeo/isolamento & purificação , Linhagem Celular , Reações Cruzadas , DNA Viral , Epitopos/imunologia , Regulação Viral da Expressão Gênica , Vetores Genéticos , Células HeLa , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Coelhos , Alinhamento de SequênciaRESUMO
De novo design of complex protein folds using solely computational means remains a significant challenge. Here, we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from GPCRs, are not found in the soluble proteome and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses reveal high thermal stability of the designs and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, standing as a proof-of-concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.
RESUMO
CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN PAM preference, with the N-terminus of Sc++, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse NNN PAMs and disease-related loci for potential therapeutic applications. In total, the unique approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.
RESUMO
CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , GenomaRESUMO
At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in DNA. However, precise mechanistic information to ameliorating Cas9 function is still missing. Here, multi-microsecond molecular dynamics, free-energy and multiscale simulations are combined with solution NMR and DNA cleavage experiments to resolve the catalytic mechanism of target DNA cleavage. We show that the conformation of an active HNH nuclease is tightly dependent on the catalytic Mg2+, unveiling its cardinal structural role. This activated Mg2+-bound HNH is consistently described through molecular simulations, solution NMR and DNA cleavage assays, revealing also that the protonation state of the catalytic H840 is strongly affected by active site mutations. Finally, ab-initio QM(DFT)/MM simulations and metadynamics establish the catalytic mechanism, showing that the catalysis is activated by H840 and completed by K866, rationalising DNA cleavage experiments. This information is critical to enhance the enzymatic function of CRISPR-Cas9 toward improved genome-editing.
RESUMO
Most known pathogenic point mutations in humans are Câ¢G to Tâ¢A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle-based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.