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1.
Toxins (Basel) ; 13(4)2021 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-33916905

RESUMEN

The CRISPR-Cas13 system based on a bacterial enzyme has been explored as a powerful new method for RNA manipulation. Due to the high efficiency and specificity of RNA editing/interference achieved by this system, it is currently being developed as a new therapeutic tool for the treatment of neurological and other diseases. However, the safety of this new generation of RNA therapies is still unclear. In this study, we constructed a vector expressing CRISPR-Cas13 under a constitutive neuron-specific promoter. CRISPR-Cas13 from Leptotrichia wadei was expressed in primary cultures of mouse cortical neurons. We found that the presence of CRISPR-Cas13 impedes the development of cultured neurons. These results show a neurotoxic action of Cas13 and call for more studies to test for and possibly mitigate the toxic effects of Cas13 enzymes in order to improve CRISPR-Cas13-based tools for RNA targeting.


Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Corteza Cerebral/enzimología , Leptotrichia/enzimología , Proyección Neuronal , Neuronas/enzimología , Animales , Proteínas Asociadas a CRISPR/genética , Células Cultivadas , Corteza Cerebral/patología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Leptotrichia/genética , Ratones , Neuronas/patología
2.
Nat Biomed Eng ; 4(12): 1140-1149, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32848209

RESUMEN

Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.


Asunto(s)
COVID-19/diagnóstico , Proteínas Asociadas a CRISPR/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/genética , SARS-CoV-2/genética , COVID-19/virología , Humanos , Leptotrichia/enzimología , Pandemias/prevención & control
3.
ACS Sens ; 4(4): 1048-1054, 2019 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-30860365

RESUMEN

Highly infectious illness caused by pathogens is endemic especially in developing nations where there is limited laboratory infrastructure and trained personnel. Rapid point-of-care (POC) serological assays with minimal sample manipulation and low cost are desired in clinical practice. In this study, we report an automated POC system for Ebola RNA detection with RNA-guided RNA endonuclease Cas13a, utilizing its collateral RNA degradation after its activation. After automated microfluidic mixing and hybridization, nonspecific cleavage products of Cas13a are immediately measured by a custom integrated fluorometer which is small in size and convenient for in-field diagnosis. Within 5 min, a detection limit of 20 pfu/mL (5.45 × 107 copies/mL) of purified Ebola RNA is achieved. This isothermal and fully solution-based diagnostic method is rapid, amplification-free, simple, and sensitive, thus establishing a key technology toward a useful POC diagnostic platform.


Asunto(s)
Sistemas CRISPR-Cas , Ebolavirus/aislamiento & purificación , Endorribonucleasas/química , Técnicas Analíticas Microfluídicas/métodos , Secuencia de Bases , Ebolavirus/genética , Fluorometría , Dispositivos Laboratorio en un Chip , Leptotrichia/enzimología , Límite de Detección , Técnicas Analíticas Microfluídicas/instrumentación , Hibridación de Ácido Nucleico , Pruebas en el Punto de Atención , ARN Viral/química , ARN Viral/genética
4.
Nucleic Acids Res ; 46(15): e90, 2018 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-29860393

RESUMEN

In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first implemented a member of the type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a), in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. This approach was shown to knock down targeted endogenous gene transcripts with different efficiencies. Second, we engineered an RNA editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human adenosine deaminase acting on RNA type 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized system parameters using a dual-fluorescence reporter and demonstrated the utility of the system in editing randomly selected endogenous gene transcripts. We further used it to restore the transposition of retrotransposon Tf1 mutants in fission yeast, providing a potential novel toolset for retrovirus manipulation and interference.


Asunto(s)
Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Leptotrichia/enzimología , Edición de ARN/genética , Ribonucleasas/genética , Schizosaccharomyces/genética , Proteínas Bacterianas/metabolismo , Regulación Fúngica de la Expresión Génica , Mutagénesis Insercional , ARN de Hongos/genética , ARN de Hongos/metabolismo , Reproducibilidad de los Resultados , Retroelementos/genética , Ribonucleasas/metabolismo , Schizosaccharomyces/metabolismo
5.
Nature ; 550(7675): 280-284, 2017 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-28976959

RESUMEN

RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.


Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edición Génica , Técnicas de Silenciamiento del Gen/métodos , Leptotrichia/enzimología , ARN/genética , ARN/metabolismo , Biocatálisis , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Línea Celular Tumoral , Supervivencia Celular , Escherichia coli/genética , Genes Reporteros/genética , Células HEK293 , Humanos , Leptotrichia/genética , Células Vegetales/metabolismo , ARN/análisis , Interferencia de ARN , Estrés Fisiológico , Especificidad por Sustrato
6.
Cell ; 168(1-2): 121-134.e12, 2017 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-28086085

RESUMEN

C2c2, the effector of type VI CRISPR-Cas systems, has two RNase activities-one for cutting its RNA target and the other for processing the CRISPR RNA (crRNA). Here, we report the structures of Leptotrichia shahii C2c2 in its crRNA-free and crRNA-bound states. While C2c2 has a bilobed structure reminiscent of all other Class 2 effectors, it also exhibits different structural characteristics. It contains the REC lobe with a Helical-1 domain and the NUC lobe with two HEPN domains. The two RNase catalytic pockets responsible for cleaving pre-crRNA and target RNA are independently located on Helical-1 and HEPN domains, respectively. crRNA binding induces significant conformational changes that are likely to stabilize crRNA binding and facilitate target RNA recognition. These structures provide important insights into the molecular mechanism of dual RNase activities of C2c2 and establish a framework for its future engineering as a RNA editing tool.


Asunto(s)
Sistemas CRISPR-Cas , Leptotrichia/química , Leptotrichia/enzimología , Ribonucleasas/química , Secuencia de Aminoácidos , Dominio Catalítico , Leptotrichia/clasificación , Leptotrichia/metabolismo , Modelos Moleculares , Mutagénesis , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN no Traducido/química , Alineación de Secuencia
7.
Nature ; 538(7624): 270-273, 2016 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-27669025

RESUMEN

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.


Asunto(s)
Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Leptotrichia/enzimología , División del ARN , ARN Bacteriano/metabolismo , Ribonucleasas/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas/genética , ARN Bacteriano/genética , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Ribonucleasas/química
9.
Science ; 353(6299): aaf5573, 2016 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-27256883

RESUMEN

The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated genes (Cas) adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the class 2 type VI CRISPR-Cas effector C2c2 and demonstrate its RNA-guided ribonuclease function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis shows that C2c2 is guided by a single CRISPR RNA and can be programmed to cleave single-stranded RNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, mutations of which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.


Asunto(s)
Inmunidad Adaptativa/genética , Proteínas Bacterianas/metabolismo , Sistemas CRISPR-Cas , Endorribonucleasas/metabolismo , Leptotrichia/enzimología , ARN Guía de Kinetoplastida/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endorribonucleasas/química , Endorribonucleasas/genética , Escherichia coli/genética , Escherichia coli/inmunología , Escherichia coli/virología , Técnicas de Silenciamiento del Gen , Sitios Genéticos , Leptotrichia/genética , Leptotrichia/inmunología , Levivirus/inmunología , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , División del ARN
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