RESUMO
CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease) defense systems have been naturally coopted for guide RNA-directed transposition on multiple occasions. In all cases, cooption occurred with diverse elements related to the bacterial transposon Tn7. Tn7 tightly controls transposition; the transposase is activated only when special targets are recognized by dedicated target-site selection proteins. Tn7 and the Tn7-like elements that coopted CRISPR-Cas systems evolved complementary targeting pathways: one that recognizes a highly conserved site in the chromosome and a second pathway that targets mobile plasmids capable of cell-to-cell transfer. Tn7 and Tn7-like elements deliver a single integration into the site they recognize and also control the orientation of the integration event, providing future potential for use as programmable gene-integration tools. Early work has shown that guide RNA-directed transposition systems can be adapted to diverse hosts, even within microbial communities, suggesting great potential for engineering these systems as powerful gene-editing tools.
Assuntos
Sistemas CRISPR-Cas , Elementos de DNA Transponíveis , RNA Guia de Sistemas CRISPR-Cas , Transposases , Elementos de DNA Transponíveis/genética , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Transposases/metabolismo , Transposases/genética , Edição de Genes/métodos , Bactérias/genética , Plasmídeos/metabolismo , Plasmídeos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente EspaçadasRESUMO
CRISPR technologies have begun to revolutionize T cell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the T cell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human T cells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR T cell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of T cell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathways to enhance CAR T cell fitness and anti-tumor activity in vitro and in vivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.
Assuntos
Engenharia Metabólica , Linfócitos T , Humanos , Perfilação da Expressão Gênica , Engenharia Metabólica/métodos , RNA , TranscriptomaRESUMO
All-RNA-mediated targeted gene integration methods, rendering reduced immunogenicity, effective deliverability with non-viral vehicles, and a low risk of random mutagenesis, are urgently needed for next-generation gene addition technologies. Naturally occurring R2 retrotransposons hold promise in this context due to their site-specific integration profile. Here, we systematically analyzed the biodiversity of R2 elements and screened several R2 orthologs capable of full-length gene insertion in mammalian cells. Robust R2 system gene integration efficiency was attained using combined donor RNA and protein engineering. Importantly, the all-RNA-delivered engineered R2 system showed effective integration activity, with efficiency over 60% in mouse embryos. Unbiased high-throughput sequencing demonstrated that the engineered R2 system exhibited high on-target integration specificity (99%). In conclusion, our study provides engineered R2 tools for applications based on hit-and-run targeted DNA integration and insights for further optimization of retrotransposon systems.
Assuntos
RNA , Retroelementos , Animais , Retroelementos/genética , Camundongos , Humanos , RNA/genética , RNA/metabolismo , Células HEK293 , Engenharia Genética/métodos , Marcação de Genes/métodosRESUMO
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/métodosRESUMO
Here, we describe an approach to correct the genetic defect in fragile X syndrome (FXS) via recruitment of endogenous repair mechanisms. A leading cause of autism spectrum disorders, FXS results from epigenetic silencing of FMR1 due to a congenital trinucleotide (CGG) repeat expansion. By investigating conditions favorable to FMR1 reactivation, we find MEK and BRAF inhibitors that induce a strong repeat contraction and full FMR1 reactivation in cellular models. We trace the mechanism to DNA demethylation and site-specific R-loops, which are necessary and sufficient for repeat contraction. A positive feedback cycle comprising demethylation, de novo FMR1 transcription, and R-loop formation results in the recruitment of endogenous DNA repair mechanisms that then drive excision of the long CGG repeat. Repeat contraction is specific to FMR1 and restores the production of FMRP protein. Our study therefore identifies a potential method of treating FXS in the future.
Assuntos
Síndrome do Cromossomo X Frágil , Expansão das Repetições de Trinucleotídeos , Humanos , Estruturas R-Loop , Metilação de DNA , Síndrome do Cromossomo X Frágil/genética , Epigênese Genética , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismoRESUMO
Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types. Using different glycoproteins in eVLPs alters their cellular tropism. Single injections of eVLPs into mice support therapeutic levels of base editing in multiple tissues, reducing serum Pcsk9 levels 78% following 63% liver editing, and partially restoring visual function in a mouse model of genetic blindness. In vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and nonviral delivery.
Assuntos
Sistemas de Liberação de Medicamentos , Engenharia Genética , Proteínas/uso terapêutico , Vírion/genética , Animais , Sequência de Bases , Cegueira/genética , Cegueira/terapia , Encéfalo/metabolismo , DNA/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Edição de Genes , Células HEK293 , Humanos , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/metabolismo , Epitélio Pigmentado da Retina/patologia , Retroviridae , Vírion/ultraestrutura , Visão OcularRESUMO
Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) is proving successful to treat several genetic diseases. HSPCs are mobilized, harvested, genetically corrected ex vivo, and infused, after the administration of toxic myeloablative conditioning to deplete the bone marrow (BM) for the modified cells. We show that mobilizers create an opportunity for seamless engraftment of exogenous cells, which effectively outcompete those mobilized, to repopulate the depleted BM. The competitive advantage results from the rescue during ex vivo culture of a detrimental impact of mobilization on HSPCs and can be further enhanced by the transient overexpression of engraftment effectors exploiting optimized mRNA-based delivery. We show the therapeutic efficacy in a mouse model of hyper IgM syndrome and further developed it in human hematochimeric mice, showing its applicability and versatility when coupled with gene transfer and editing strategies. Overall, our findings provide a potentially valuable strategy paving the way to broader and safer use of HSPC-GT.
Assuntos
Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Animais , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas , Humanos , CamundongosRESUMO
Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.
Assuntos
Alelos , Proteína 9 Associada à CRISPR/metabolismo , Cromossomos Humanos/genética , Embrião de Mamíferos/metabolismo , Animais , Sequência de Bases , Blastocisto/metabolismo , Ciclo Celular/genética , Linhagem Celular , Deleção Cromossômica , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Implantação do Embrião/genética , Proteínas do Olho/genética , Fertilização , Edição de Genes , Rearranjo Gênico/genética , Loci Gênicos , Genoma Humano , Genótipo , Heterozigoto , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mutação INDEL/genética , Camundongos , Mitose , Fases de Leitura Aberta/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
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 , SoftwareRESUMO
Class 2 CRISPR-Cas systems endow microbes with diverse mechanisms for adaptive immunity. Here, we analyzed prokaryotic genome and metagenome sequences to identify an uncharacterized family of RNA-guided, RNA-targeting CRISPR systems that we classify as type VI-D. Biochemical characterization and protein engineering of seven distinct orthologs generated a ribonuclease effector derived from Ruminococcus flavefaciens XPD3002 (CasRx) with robust activity in human cells. CasRx-mediated knockdown exhibits high efficiency and specificity relative to RNA interference across diverse endogenous transcripts. As one of the most compact single-effector Cas enzymes, CasRx can also be flexibly packaged into adeno-associated virus. We target virally encoded, catalytically inactive CasRx to cis elements of pre-mRNA to manipulate alternative splicing, alleviating dysregulated tau isoform ratios in a neuronal model of frontotemporal dementia. Our results present CasRx as a programmable RNA-binding module for efficient targeting of cellular RNA, enabling a general platform for transcriptome engineering and future therapeutic development.
Assuntos
Sistemas CRISPR-Cas , Biologia Computacional/métodos , Engenharia Genética/métodos , Engenharia de Proteínas/métodos , RNA/análise , Processamento Alternativo , Animais , Proteínas de Bactérias/metabolismo , Diferenciação Celular , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Lentivirus/genética , Camundongos , Interferência de RNA , RNA Guia de Cinetoplastídeos/genética , Ruminococcus , Análise de Sequência de RNA , TranscriptomaRESUMO
The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.
Assuntos
Células-Tronco Hematopoéticas/citologia , Imunoterapia/métodos , Leucemia Mieloide Aguda/terapia , RNA Guia de Cinetoplastídeos/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Linfócitos T/imunologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Linhagem da Célula , Eletroporação , Feminino , Hematopoese , Humanos , Leucemia Mieloide Aguda/imunologia , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Transplante de Neoplasias , Espécies Reativas de Oxigênio , Linfócitos T/citologiaRESUMO
Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2ß2) disorders, sickle cell disease, and ß-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to ß- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.
Assuntos
Proteínas de Transporte/metabolismo , Hemoglobina Fetal/genética , Proteínas Nucleares/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Linhagem Celular , Cromatina/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células Eritroides/citologia , Células Eritroides/metabolismo , Edição de Genes , Humanos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras , Dedos de Zinco/genética , Globinas beta/genética , Talassemia beta/genética , Talassemia beta/patologia , gama-Globinas/genéticaRESUMO
CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9.
Assuntos
Sistemas CRISPR-Cas , Endonucleases/antagonistas & inibidores , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , Evolução Molecular , Células HEK293 , Humanos , Domínios Proteicos , Alinhamento de SequênciaRESUMO
Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.
Assuntos
Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/genética , Animais , Encéfalo/fisiologia , Cromossomos Humanos X , Ritmo Circadiano , Modelos Animais de Doenças , Eletrocardiografia , Feminino , Edição de Genes , Humanos , Macaca fascicularis , Imageamento por Ressonância Magnética , Masculino , Mutação , Dor , Síndrome de Rett/fisiopatologia , Sono , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , TranscriptomaRESUMO
Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9.
Assuntos
Bacteriófagos/metabolismo , Sistemas CRISPR-Cas , Endonucleases/antagonistas & inibidores , Engenharia Genética , Listeria monocytogenes/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteína 9 Associada à CRISPR , Escherichia coli , Células HEK293 , Humanos , Listeria monocytogenes/imunologia , Listeria monocytogenes/virologia , PrófagosRESUMO
Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases. VIDEO ABSTRACT.
Assuntos
Sistemas CRISPR-Cas , Epigênese Genética , Marcação de Genes/métodos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Utrofina/genética , Animais , Sequência de Bases , Modelos Animais de Doenças , Distrofina/genética , Interleucina-10/genética , Proteínas Klotho , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Ativação TranscricionalRESUMO
Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.
Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/imunologia , Bacteriófagos/genética , Bacteriófagos/imunologia , Proteínas Associadas a CRISPR/imunologia , Proteínas Associadas a CRISPR/ultraestrutura , DNA Viral/química , Modelos Químicos , Modelos Moleculares , Complexos Multiproteicos/química , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/ultraestruturaRESUMO
The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.
Assuntos
Acidaminococcus , Proteínas de Bactérias , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Domínio Catalítico , Microscopia Crioeletrônica , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Acidaminococcus/enzimologia , Acidaminococcus/genética , Acidaminococcus/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Estruturas R-Loop/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/química , RNA Guia de Sistemas CRISPR-Cas/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , Modelos Moleculares , Domínios Proteicos , Relação Estrutura-Atividade , Ligação ProteicaRESUMO
CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Genoma , DNA/genética , Pinças ÓpticasRESUMO
CD4+ T cells share common developmental pathways with CD8+ T cells, and upon maturation, CD4+ T conventional T (Tconv) cells lack phenotypic markers that distinguish these cells from FoxP3+ T regulatory cells. We developed a tamoxifen-inducible ThPOKCreERT2.hCD2 line with Frt sites inserted on either side of the CreERT2-hCD2 cassette, and a Foxp3Ametrine-FlpO strain, expressing Ametrine and FlpO in Foxp3+ cells. Breeding these mice resulted in a CD4conviCreERT2-hCD2 line that allows for the specific manipulation of a gene in CD4+ Tconv cells. As FlpO removes the CreERT2-hCD2 cassette, CD4+ Treg cells are spared from Cre activity, which we refer to as allele conditioning. Comparison with an E8IiCreERT2.GFP mouse that enables inducible targeting of CD8+ T cells, and deletion of two inhibitory receptors, PD-1 and LAG-3, in a melanoma model, support the fidelity of these lines. These engineered mouse strains present a resource for the temporal manipulation of genes in CD4+ T cells and CD4+ Tconv cells.