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1.
Annu Rev Biochem ; 93(1): 139-161, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38598855

RESUMEN

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.


Asunto(s)
Sistemas CRISPR-Cas , Elementos Transponibles de ADN , ARN Guía de Sistemas CRISPR-Cas , Transposasas , Elementos Transponibles de ADN/genética , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , Transposasas/metabolismo , Transposasas/genética , Edición Génica/métodos , Bacterias/genética , Plásmidos/metabolismo , Plásmidos/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas
2.
Cell ; 187(10): 2411-2427.e25, 2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38608704

RESUMEN

We set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing, a precise genome engineering tool. Using a highly sensitive method for mapping the genomic locations of randomly integrated reporters, we discover massive position effects, exemplified by editing efficiencies ranging from ∼0% to 94% for an identical target site and edit. Position effects on prime editing efficiency are well predicted by chromatin marks, e.g., positively by H3K79me2 and negatively by H3K9me3. Next, we developed a multiplex perturbational framework to assess the interaction of trans-acting factors with the cis-chromatin environment on editing outcomes. Applying this framework to DNA repair factors, we identify HLTF as a context-dependent repressor of prime editing. Finally, several lines of evidence suggest that active transcriptional elongation enhances prime editing. Consistent with this, we show we can robustly decrease or increase the efficiency of prime editing by preceding it with CRISPR-mediated silencing or activation, respectively.


Asunto(s)
Sistemas CRISPR-Cas , Cromatina , Epigénesis Genética , Edición Génica , Humanos , Cromatina/metabolismo , Cromatina/genética , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Histonas/metabolismo , Factores de Transcripción/metabolismo , Código de Histonas
3.
Cell ; 187(15): 3936-3952.e19, 2024 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-38936359

RESUMEN

Duplication is a foundation of molecular evolution and a driver of genomic and complex diseases. Here, we develop a genome editing tool named Amplification Editing (AE) that enables programmable DNA duplication with precision at chromosomal scale. AE can duplicate human genomes ranging from 20 bp to 100 Mb, a size comparable to human chromosomes. AE exhibits activity across various cell types, encompassing diploid, haploid, and primary cells. AE exhibited up to 73.0% efficiency for 1 Mb and 3.4% for 100 Mb duplications, respectively. Whole-genome sequencing and deep sequencing of the junctions of edited sequences confirm the precision of duplication. AE can create chromosomal microduplications within disease-relevant regions in embryonic stem cells, indicating its potential for generating cellular and animal models. AE is a precise and efficient tool for chromosomal engineering and DNA duplication, broadening the landscape of precision genome editing from an individual genetic locus to the chromosomal scale.


Asunto(s)
Duplicación de Gen , Edición Génica , Genoma Humano , Humanos , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , ADN/genética , Animales , Células Madre Embrionarias/metabolismo , Cromosomas Humanos/genética
4.
Cell ; 187(20): 5719-5734.e19, 2024 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-39299233

RESUMEN

Pathogenic variants in RAD51C confer an elevated risk of breast and ovarian cancer, while individuals homozygous for specific RAD51C alleles may develop Fanconi anemia. Using saturation genome editing (SGE), we functionally assess 9,188 unique variants, including >99.5% of all possible coding sequence single-nucleotide alterations. By computing changes in variant abundance and Gaussian mixture modeling (GMM), we functionally classify 3,094 variants to be disruptive and use clinical truth sets to reveal an accuracy/concordance of variant classification >99.9%. Cell fitness was the primary assay readout allowing us to observe a phenomenon where specific missense variants exhibit distinct depletion kinetics potentially suggesting that they represent hypomorphic alleles. We further explored our exhaustive functional map, revealing critical residues on the RAD51C structure and resolving variants found in cancer-segregating kindred. Furthermore, through interrogation of UK Biobank and a large multi-center ovarian cancer cohort, we find significant associations between SGE-depleted variants and cancer diagnoses.


Asunto(s)
Proteínas de Unión al ADN , Edición Génica , Neoplasias Ováricas , Humanos , Femenino , Edición Génica/métodos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Neoplasias Ováricas/genética , Neoplasias de la Mama/genética , Alelos , Sistemas CRISPR-Cas/genética
5.
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
6.
Cell ; 186(10): 2256-2272.e23, 2023 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-37119812

RESUMEN

Applications of prime editing are often limited due to insufficient efficiencies, and it can require substantial time and resources to determine the most efficient pegRNAs and prime editors (PEs) to generate a desired edit under various experimental conditions. Here, we evaluated prime editing efficiencies for a total of 338,996 pairs of pegRNAs including 3,979 epegRNAs and target sequences in an error-free manner. These datasets enabled a systematic determination of factors affecting prime editing efficiencies. Then, we developed computational models, named DeepPrime and DeepPrime-FT, that can predict prime editing efficiencies for eight prime editing systems in seven cell types for all possible types of editing of up to 3 base pairs. We also extensively profiled the prime editing efficiencies at mismatched targets and developed a computational model predicting editing efficiencies at such targets. These computational models, together with our improved knowledge about prime editing efficiency determinants, will greatly facilitate prime editing applications.


Asunto(s)
Simulación por Computador , Edición Génica , ARN Guía de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas , Edición Génica/métodos , Conocimiento , ARN Guía de Sistemas CRISPR-Cas/química , Especificidad de Órganos , Conjuntos de Datos como Asunto
7.
Cell ; 186(21): 4567-4582.e20, 2023 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-37794590

RESUMEN

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


Asunto(s)
Sistemas CRISPR-Cas , Aberraciones Cromosómicas , Edición Génica , Linfocitos T , Humanos , Cromosomas , Sistemas CRISPR-Cas/genética , Daño del ADN , Edición Génica/métodos , Ensayos Clínicos como Asunto
8.
Cell ; 185(15): 2806-2827, 2022 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-35798006

RESUMEN

In vivo gene editing therapies offer the potential to treat the root causes of many genetic diseases. Realizing the promise of therapeutic in vivo gene editing requires the ability to safely and efficiently deliver gene editing agents to relevant organs and tissues in vivo. Here, we review current delivery technologies that have been used to enable therapeutic in vivo gene editing, including viral vectors, lipid nanoparticles, and virus-like particles. Since no single delivery modality is likely to be appropriate for every possible application, we compare the benefits and drawbacks of each method and highlight opportunities for future improvements.


Asunto(s)
Edición Génica , Nanopartículas , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Terapia Genética/métodos , Vectores Genéticos , Liposomas
9.
Nat Rev Mol Cell Biol ; 25(6): 464-487, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38308006

RESUMEN

Our ability to edit genomes lags behind our capacity to sequence them, but the growing understanding of CRISPR biology and its application to genome, epigenome and transcriptome engineering is narrowing this gap. In this Review, we discuss recent developments of various CRISPR-based systems that can transiently or permanently modify the genome and the transcriptome. The discovery of further CRISPR enzymes and systems through functional metagenomics has meaningfully broadened the applicability of CRISPR-based editing. Engineered Cas variants offer diverse capabilities such as base editing, prime editing, gene insertion and gene regulation, thereby providing a panoply of tools for the scientific community. We highlight the strengths and weaknesses of current CRISPR tools, considering their efficiency, precision, specificity, reliance on cellular DNA repair mechanisms and their applications in both fundamental biology and therapeutics. Finally, we discuss ongoing clinical trials that illustrate the potential impact of CRISPR systems on human health.


Asunto(s)
Sistemas CRISPR-Cas , Epigenoma , Edición Génica , Transcriptoma , Humanos , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Epigenoma/genética , Animales , Transcriptoma/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genoma/genética
10.
Annu Rev Biochem ; 89: 77-101, 2020 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-32569517

RESUMEN

DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.


Asunto(s)
ADN/genética , Edición Génica/métodos , Técnicas de Transferencia de Gen , Genes Sintéticos , Ingeniería Genética/métodos , Genoma , Sistemas CRISPR-Cas , ADN/química , ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Oligonucleótidos/síntesis química , Oligonucleótidos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Poliovirus/genética , Poliovirus/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esferoplastos/genética , Esferoplastos/metabolismo
11.
Annu Rev Biochem ; 89: 309-332, 2020 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-32186918

RESUMEN

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.


Asunto(s)
Sistemas CRISPR-Cas/efectos de los fármacos , Edición Génica/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas Virales/genética , Virus/genética , Archaea/genética , Archaea/inmunología , Archaea/virología , Bacterias/genética , Bacterias/inmunología , Bacterias/virología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Coevolución Biológica , Proteínas Asociadas a CRISPR/antagonistas & inhibidores , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , ADN/antagonistas & inhibidores , ADN/química , ADN/genética , ADN/metabolismo , División del ADN/efectos de los fármacos , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Humanos , Modelos Moleculares , Familia de Multigenes , Unión Proteica , Multimerización de Proteína/efectos de los fármacos , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Proteínas Virales/farmacología , Virus/metabolismo , Virus/patogenicidad
12.
Cell ; 181(1): 136-150, 2020 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-32243786

RESUMEN

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments.


Asunto(s)
Sistemas CRISPR-Cas , Dependovirus/genética , Edición Génica/métodos , Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Animales , Humanos
13.
Cell ; 182(2): 463-480.e30, 2020 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-32533916

RESUMEN

Although base editors are widely used to install targeted point mutations, the factors that determine base editing outcomes are not well understood. We characterized sequence-activity relationships of 11 cytosine and adenine base editors (CBEs and ABEs) on 38,538 genomically integrated targets in mammalian cells and used the resulting outcomes to train BE-Hive, a machine learning model that accurately predicts base editing genotypic outcomes (R ≈ 0.9) and efficiency (R ≈ 0.7). We corrected 3,388 disease-associated SNVs with ≥90% precision, including 675 alleles with bystander nucleotides that BE-Hive correctly predicted would not be edited. We discovered determinants of previously unpredictable C-to-G, or C-to-A editing and used these discoveries to correct coding sequences of 174 pathogenic transversion SNVs with ≥90% precision. Finally, we used insights from BE-Hive to engineer novel CBE variants that modulate editing outcomes. These discoveries illuminate base editing, enable editing at previously intractable targets, and provide new base editors with improved editing capabilities.


Asunto(s)
Edición Génica/métodos , Aprendizaje Automático , Animales , Biblioteca de Genes , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Mutación Puntual , ARN Guía de Kinetoplastida/metabolismo
14.
Cell ; 181(5): 955-960, 2020 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-32470403

RESUMEN

The first clinical studies utilizing RNA-guided endonucleases (RGENs) to therapeutically edit RNA and DNA in cancer patients were recently published. These groundbreaking technological advances promise to revolutionize genetic therapy and, as I discuss, represent the culmination of decades of innovative work to engineer RGENs for such editing applications.


Asunto(s)
Edición Génica/métodos , Edición Génica/tendencias , Edición de ARN/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN/genética , Endonucleasas/metabolismo , Mutación , ARN/genética , Edición de ARN/fisiología , ARN Catalítico/genética , ARN Guía de Kinetoplastida/genética
15.
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
16.
Cell ; 179(7): 1448-1450, 2019 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-31835025

RESUMEN

Many targeted base transversions, insertions, and deletions remain challenging due to the lack of precise and efficient genome editing technologies. Recently, Anzalone et al. reported a versatile approach to achieve all types of genome edits, shedding new light on correcting most genetic variants associated with diseases.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Animales , Terapia Genética/métodos , Humanos
17.
Cell ; 176(1-2): 254-267.e16, 2019 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-30633905

RESUMEN

The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.


Asunto(s)
Sistemas CRISPR-Cas/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/fisiología , Edición Génica/métodos , Proteínas Asociadas a CRISPR/química , ADN/química , Genoma , Modelos Moleculares , ARN/química , ARN Guía de Kinetoplastida/genética
18.
Cell ; 178(1): 122-134.e12, 2019 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-31230714

RESUMEN

Epitranscriptomic regulation controls information flow through the central dogma and provides unique opportunities for manipulating cells at the RNA level. However, both fundamental studies and potential translational applications are impeded by a lack of methods to target specific RNAs with effector proteins. Here, we present CRISPR-Cas-inspired RNA targeting system (CIRTS), a protein engineering strategy for constructing programmable RNA control elements. We show that CIRTS is a simple and generalizable approach to deliver a range of effector proteins, including nucleases, degradation machinery, translational activators, and base editors to target transcripts. We further demonstrate that CIRTS is not only smaller than naturally occurring CRISPR-Cas programmable RNA binding systems but can also be built entirely from human protein parts. CIRTS provides a platform to probe fundamental RNA regulatory processes, and the human-derived nature of CIRTS provides a potential strategy to avoid immune issues when applied to epitranscriptome-modulating therapies.


Asunto(s)
Edición Génica/métodos , Ingeniería de Proteínas/métodos , ARN Guía de Kinetoplastida/metabolismo , ARN/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismo , Sistemas CRISPR-Cas/genética , Escherichia coli/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Biosíntesis de Proteínas , Proteolisis , ARN Interferente Pequeño , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Transfección
19.
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
20.
Cell ; 175(3): 615-632, 2018 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-30340033

RESUMEN

The derivation of human embryonic stem cells (hESCs) and the stunning discovery that somatic cells can be reprogrammed into human induced pluripotent stem cells (hiPSCs) holds the promise to revolutionize biomedical research and regenerative medicine. In this Review, we focus on disorders of the central nervous system and explore how advances in human pluripotent stem cells (hPSCs) coincide with evolutions in genome engineering and genomic technologies to provide realistic opportunities to tackle some of the most devastating complex disorders.


Asunto(s)
Enfermedades del Sistema Nervioso Central/terapia , Edición Génica/métodos , Trasplante de Células Madre/métodos , Investigación Biomédica Traslacional/métodos , Animales , Enfermedades del Sistema Nervioso Central/genética , Humanos
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