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1.
Cell ; 187(13): 3249-3261.e14, 2024 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-38781968

RESUMEN

Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.


Asunto(s)
Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Microscopía por Crioelectrón , ADN , Edición Génica , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , ADN/metabolismo , ADN/genética , Edición Génica/métodos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Células HEK293 , Dominios Proteicos , Genoma Humano , Modelos Moleculares , Estructura Terciaria de Proteína , Conformación de Ácido Nucleico , Biocatálisis , Magnesio/química , Magnesio/metabolismo
2.
Annu Rev Biochem ; 92: 43-79, 2023 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-37018843

RESUMEN

DNA-editing enzymes perform chemical reactions on DNA nucleobases. These reactions can change the genetic identity of the modified base or modulate gene expression. Interest in DNA-editing enzymes has burgeoned in recent years due to the advent of clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) systems, which can be used to direct their DNA-editing activity to specific genomic loci of interest. In this review, we showcase DNA-editing enzymes that have been repurposed or redesigned and developed into programmable base editors. These include deaminases, glycosylases, methyltransferases, and demethylases. We highlight the astounding degree to which these enzymes have been redesigned, evolved, and refined and present these collective engineering efforts as a paragon for future efforts to repurpose and engineer other families of enzymes. Collectively, base editors derived from these DNA-editing enzymes facilitate programmable point mutation introduction and gene expression modulation by targeted chemical modification of nucleobases.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Proteína 9 Asociada a CRISPR/genética , Genoma , ADN/genética , ADN/metabolismo
3.
Cell ; 184(22): 5653-5669.e25, 2021 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-34672952

RESUMEN

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.


Asunto(s)
Roturas del ADN de Doble Cadena , Genómica , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular , Análisis por Conglomerados , Reparación del ADN/genética , Edición Génica , Regulación de la Expresión Génica , Genoma Humano , Humanos , Fenotipo , ARN Guía de Kinetoplastida/metabolismo , Reproducibilidad de los Resultados
4.
Cell ; 184(4): 1047-1063.e23, 2021 02 18.
Artículo en Inglés | MEDLINE | ID: mdl-33539780

RESUMEN

DNA has not been utilized to record temporal information, although DNA has been used to record biological information and to compute mathematical problems. Here, we found that indel generation by Cas9 and guide RNA can occur at steady rates, in contrast to typical dynamic biological reactions, and the accumulated indel frequency can be a function of time. By measuring indel frequencies, we developed a method for recording and measuring absolute time periods over hours to weeks in mammalian cells. These time-recordings were conducted in several cell types, with different promoters and delivery vectors for Cas9, and in both cultured cells and cells of living mice. As applications, we recorded the duration of chemical exposure and the lengths of elapsed time since the onset of biological events (e.g., heat exposure and inflammation). We propose that our systems could serve as synthetic "DNA clocks."


Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Animales , Secuencia de Bases , Microambiente Celular , Simulación por Computador , Células HEK293 , Semivida , Humanos , Mutación INDEL/genética , Inflamación/patología , Integrasas/metabolismo , Masculino , Ratones Desnudos , Regiones Promotoras Genéticas/genética , ARN Guía de Kinetoplastida/genética , Reproducibilidad de los Resultados , Factores de Tiempo
5.
Cell ; 184(12): 3267-3280.e18, 2021 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-34043941

RESUMEN

Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.


Asunto(s)
Reparación del ADN/genética , Conversión Génica , Recombinasa Rad51/metabolismo , Alelos , Animales , Secuencia de Bases , Proteína 9 Asociada a CRISPR/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromosomas de los Mamíferos/genética , Roturas del ADN de Doble Cadena , Embrión de Mamíferos , Femenino , Sitios Genéticos , Recombinación Homóloga/genética , Homocigoto , Humanos , Mutación INDEL/genética , Ratones Endogámicos C57BL , Mosaicismo , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleótido Simple/genética , Ribonucleoproteínas/metabolismo , Cigoto/metabolismo
6.
Cell ; 184(3): 675-688.e19, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33421369

RESUMEN

CRISPR-Cas systems provide prokaryotes with acquired immunity against viruses and plasmids, but how these systems are regulated to prevent autoimmunity is poorly understood. Here, we show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA (tracr-L) folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas). Further, we demonstrate that Pcas serves as a critical regulatory node. De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses; however, heightened immunity comes at the cost of increased autoimmune toxicity. Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.


Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Expresión Génica , Homeostasis/genética , ARN Guía de Kinetoplastida/genética , Autoinmunidad/genética , Secuencia de Bases , Secuencia Conservada , Regulación hacia Abajo/genética , Modelos Genéticos , Mutación/genética , Operón/genética , Regiones Promotoras Genéticas/genética , Streptococcus pyogenes/genética , Estrés Fisiológico/genética , Transcripción Genética , Activación Transcripcional/genética
7.
Cell ; 184(21): 5357-5374.e22, 2021 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-34582788

RESUMEN

Despite remarkable clinical efficacy of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits for triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that deletion of the E3 ubiquitin ligase Cop1 in cancer cells decreases secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, enhances anti-tumor immunity, and strengthens ICB response. Transcriptomics, epigenomics, and proteomics analyses revealed that Cop1 functions through proteasomal degradation of the C/ebpδ protein. The Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. In addition, deletion of the E3 ubiquitin ligase Cop1 in cancer cells stabilizes C/ebpδ to suppress expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy in TNBC by regulating chemokine secretion and macrophage infiltration in the tumor microenvironment.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Inmunoterapia , Macrófagos/enzimología , Neoplasias/inmunología , Neoplasias/terapia , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular Tumoral , Quimiocinas/metabolismo , Quimiotaxis , Modelos Animales de Enfermedad , Biblioteca de Genes , Humanos , Evasión Inmune , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteolisis , Especificidad por Sustrato , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/terapia
8.
Cell ; 183(6): 1650-1664.e15, 2020 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-33125898

RESUMEN

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.


Asunto(s)
Alelos , Proteína 9 Asociada a CRISPR/metabolismo , Cromosomas Humanos/genética , Embrión de Mamíferos/metabolismo , Animales , Secuencia de Bases , Blastocisto/metabolismo , Ciclo Celular/genética , Línea Celular , Deleción Cromosómica , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Implantación del Embrión/genética , Proteínas del Ojo/genética , Fertilización , Edición Génica , Reordenamiento Génico/genética , Sitios Genéticos , Genoma Humano , Genotipo , Heterocigoto , Células Madre Embrionarias Humanas/metabolismo , Humanos , Mutación INDEL/genética , Ratones , Mitosis , Sistemas de Lectura Abierta/genética , Polimorfismo de Nucleótido Simple/genética
9.
Annu Rev Biochem ; 88: 191-220, 2019 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-30883196

RESUMEN

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.


Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica/métodos , Ingeniería de Proteínas/métodos , ARN Guía de Kinetoplastida/genética , Desaminasas APOBEC/genética , Desaminasas APOBEC/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Artefactos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Transferencia de Gen , Genoma Humano , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Programas Informáticos
10.
Cell ; 177(4): 1067-1079.e19, 2019 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-31051099

RESUMEN

The precise control of CRISPR-Cas9 activity is required for a number of genome engineering technologies. Here, we report a generalizable platform that provided the first synthetic small-molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) that weigh <500 Da and are cell permeable, reversible, and stable under physiological conditions. We developed a suite of high-throughput assays for SpCas9 functions, including a primary screening assay for SpCas9 binding to the protospacer adjacent motif, and used these assays to screen a structurally diverse collection of natural-product-like small molecules to ultimately identify compounds that disrupt the SpCas9-DNA interaction. Using these synthetic anti-CRISPR small molecules, we demonstrated dose and temporal control of SpCas9 and catalytically impaired SpCas9 technologies, including transcription activation, and identified a pharmacophore for SpCas9 inhibition using structure-activity relationships. These studies establish a platform for rapidly identifying synthetic, miniature, cell-permeable, and reversible inhibitors against both SpCas9 and next-generation CRISPR-associated nucleases.


Asunto(s)
Proteína 9 Asociada a CRISPR/antagonistas & inhibidores , Sistemas CRISPR-Cas/fisiología , Ensayos Analíticos de Alto Rendimiento/métodos , Proteína 9 Asociada a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , ADN/metabolismo , Endonucleasas/metabolismo , Edición Génica/métodos , Genoma , Bibliotecas de Moléculas Pequeñas , Streptococcus pyogenes/genética , Especificidad por Sustrato
11.
Cell ; 175(7): 1958-1971.e15, 2018 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-30449619

RESUMEN

Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, single guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA sequencing (RNA-seq) revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.


Asunto(s)
Sistemas CRISPR-Cas , Genoma Humano , Linfocitos T/inmunología , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/inmunología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Inactivación de Genes , Estudio de Asociación del Genoma Completo , Humanos , Linfocitos T/citología
12.
Cell ; 172(5): 979-992.e6, 2018 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-29456084

RESUMEN

Fragile X syndrome (FXS), the most common genetic form of intellectual disability in males, is caused by silencing of the FMR1 gene associated with hypermethylation of the CGG expansion mutation in the 5' UTR of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/single guide RNA (sgRNA) switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state, restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation-edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish that demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.


Asunto(s)
Metilación de ADN/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Edición Génica , Neuronas/patología , Animales , Proteína 9 Asociada a CRISPR/metabolismo , Epigénesis Genética , Células HEK293 , Heterocromatina/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cinética , Masculino , Ratones , Neuronas/metabolismo , Fenotipo , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/metabolismo , Expansión de Repetición de Trinucleótido/genética
13.
Cell ; 168(1-2): 150-158.e10, 2017 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-28041849

RESUMEN

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.


Asunto(s)
Bacteriófagos/metabolismo , Sistemas CRISPR-Cas , Endonucleasas/antagonistas & inhibidores , Ingeniería Genética , Listeria monocytogenes/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteína 9 Asociada a CRISPR , Escherichia coli , Células HEK293 , Humanos , Listeria monocytogenes/inmunología , Listeria monocytogenes/virología , Profagos
14.
Mol Cell ; 84(8): 1585-1600.e7, 2024 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-38479385

RESUMEN

Myriad physiological and pathogenic processes are governed by protein levels and modifications. Controlled protein activity perturbation is essential to studying protein function in cells and animals. Based on Trim-Away technology, we screened for truncation variants of E3 ubiquitinase Trim21 with elevated efficiency (ΔTrim21) and developed multiple ΔTrim21-based targeted protein-degradation systems (ΔTrim-TPD) that can be transfected into host cells. Three ΔTrim-TPD variants are developed to enable chemical and light-triggered programmable activation of TPD in cells and animals. Specifically, we used ΔTrim-TPD for (1) red-light-triggered inhibition of HSV-1 virus proliferation by degrading the packaging protein gD, (2) for chemical-triggered control of the activity of Cas9/dCas9 protein for gene editing, and (3) for blue-light-triggered degradation of two tumor-associated proteins for spatiotemporal inhibition of melanoma tumor growth in mice. Our study demonstrates that multiple ΔTrim21-based controllable TPD systems provide powerful tools for basic biology research and highlight their potential biomedical applications.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Ratones , Animales , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Proteínas/metabolismo , Proteolisis , Mamíferos/metabolismo
15.
Mol Cell ; 84(12): 2221-2222, 2024 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-38906114

RESUMEN

In their recent structural work, Eggers et al.1 rationalize how key mutations in the WED domain of the compact and thermostable Geobacillus stearothermophilus Cas9 bolster its editing efficiency in mammalian cells, and they use these insights to rationally improve another Cas9.


Asunto(s)
Proteína 9 Asociada a CRISPR , Edición Génica , Edición Génica/métodos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genética , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/enzimología , Sistemas CRISPR-Cas , Humanos , Mutación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Animales
16.
Annu Rev Biochem ; 85: 227-64, 2016 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-27145843

RESUMEN

The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.


Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Reparación del ADN , Endonucleasas/genética , Edición Génica/métodos , ARN Guía de Kinetoplastida/genética , Proteínas Bacterianas/metabolismo , Emparejamiento Base , Proteína 9 Asociada a CRISPR , División del ADN , Endonucleasas/metabolismo , Epigénesis Genética , Marcación de Gen , Genoma Humano , Humanos , Imagen Molecular , Ingeniería de Proteínas , Estructura Secundaria de Proteína , ARN Guía de Kinetoplastida/metabolismo
17.
Cell ; 165(6): 1295-1297, 2016 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-27259138
18.
Cell ; 166(5): 1188-1197.e9, 2016 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-27523609

RESUMEN

Telomerase maintains genome integrity by adding repetitive DNA sequences to the chromosome ends in actively dividing cells, including 90% of all cancer cells. Recruitment of human telomerase to telomeres occurs during S-phase of the cell cycle, but the molecular mechanism of the process is only partially understood. Here, we use CRISPR genome editing and single-molecule imaging to track telomerase trafficking in nuclei of living human cells. We demonstrate that telomerase uses three-dimensional diffusion to search for telomeres, probing each telomere thousands of times each S-phase but only rarely forming a stable association. Both the transient and stable association events depend on the direct interaction of the telomerase protein TERT with the telomeric protein TPP1. Our results reveal that telomerase recruitment to telomeres is driven by dynamic interactions between the rapidly diffusing telomerase and the chromosome end.


Asunto(s)
Telomerasa/metabolismo , Telómero/enzimología , Transporte Activo de Núcleo Celular , Proteínas Bacterianas , Proteína 9 Asociada a CRISPR , Línea Celular , Núcleo Celular/enzimología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cuerpos Enrollados/enzimología , Endonucleasas , Edición Génica , Genoma Humano , Células HeLa , Humanos , Imagenología Tridimensional , Dominios Proteicos , Fase S , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Complejo Shelterina , Telomerasa/química , Telómero/química , Homeostasis del Telómero , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismo
19.
Cell ; 164(5): 950-61, 2016 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-26875867

RESUMEN

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox.


Asunto(s)
Proteínas Bacterianas/química , Sistemas CRISPR-Cas , Endonucleasas/química , Francisella/enzimología , Ingeniería Genética/métodos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Blastocisto/metabolismo , Proteína 9 Asociada a CRISPR , Cristalografía por Rayos X , Embrión de Mamíferos/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Ratones , Microinyecciones/métodos , Modelos Moleculares , ARN Guía de Kinetoplastida/genética
20.
Cell ; 167(1): 233-247.e17, 2016 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-27662091

RESUMEN

Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Edición Génica/métodos , Proteínas Proto-Oncogénicas/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Factor de Unión a CCCTC , Proteína 9 Asociada a CRISPR , Línea Celular , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Elementos de Facilitación Genéticos , Genoma , Ratones , Proteína MioD/metabolismo , Neuronas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo
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