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1.
Mol Cell ; 65(4): 618-630.e7, 2017 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-28065598

RESUMO

CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 and Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Escherichia coli/enzimologia , Edição de Genes/métodos , Interferência de RNA , RNA Bacteriano/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Ribonucleases/metabolismo , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Biologia Computacional , Mineração de Dados , Bases de Dados Genéticas , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , RNA Guia de Cinetoplastídeos/genética , Ribonucleases/genética
2.
Nature ; 550(7675): 280-284, 2017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-28976959

RESUMO

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.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Silenciamento de Genes/métodos , Leptotrichia/enzimologia , RNA/genética , RNA/metabolismo , Biocatálise , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Escherichia coli/genética , Genes Reporter/genética , Células HEK293 , Humanos , Leptotrichia/genética , Células Vegetais/metabolismo , RNA/análise , Interferência de RNA , Estresse Fisiológico , Especificidade por Substrato
3.
bioRxiv ; 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38464325

RESUMO

Prediction of RNA structure from sequence remains an unsolved problem, and progress has been slowed by a paucity of experimental data. Here, we present Ribonanza, a dataset of chemical mapping measurements on two million diverse RNA sequences collected through Eterna and other crowdsourced initiatives. Ribonanza measurements enabled solicitation, training, and prospective evaluation of diverse deep neural networks through a Kaggle challenge, followed by distillation into a single, self-contained model called RibonanzaNet. When fine tuned on auxiliary datasets, RibonanzaNet achieves state-of-the-art performance in modeling experimental sequence dropout, RNA hydrolytic degradation, and RNA secondary structure, with implications for modeling RNA tertiary structure.

4.
Science ; 365(6451): 382-386, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31296651

RESUMO

Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine (A-to-I) RNA editing approach by fusing catalytically inactivate RNA-targeting CRISPR-Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Here, we report a cytidine-to-uridine (C-to-U) RNA editor, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), by directly evolving ADAR2 into a cytidine deaminase. RESCUE doubles the number of mutations targetable by RNA editing and enables modulation of phosphosignaling-relevant residues. We apply RESCUE to drive ß-catenin activation and cellular growth. Furthermore, RESCUE retains A-to-I editing activity, enabling multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs.


Assuntos
Adenosina Desaminase/genética , Citidina/genética , Citosina Desaminase/genética , Engenharia de Proteínas/métodos , Edição de RNA , Proteínas de Ligação a RNA/genética , Uridina/genética , Adenosina/genética , Adenosina Desaminase/química , Citosina Desaminase/química , Células HEK293 , Humanos , Inosina/genética , Domínios Proteicos , Proteínas de Ligação a RNA/química , beta Catenina/química , beta Catenina/genética , beta Catenina/metabolismo
5.
Science ; 358(6366): 1019-1027, 2017 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-29070703

RESUMO

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Endonucleases/metabolismo , Técnicas de Silenciamento de Genes , Edição de RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Biotecnologia , Diabetes Insípido Nefrogênico/genética , Diabetes Insípido Nefrogênico/terapia , Endonucleases/classificação , Endonucleases/genética , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Terapia Genética , Células HEK293 , Humanos , Mutagênese , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
6.
Nat Biotechnol ; 35(8): 789-792, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28581492

RESUMO

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.


Assuntos
Proteínas de Bactérias/genética , Endonucleases/genética , Engenharia Genética/métodos , Variação Genética/genética , Mutagênese Sítio-Dirigida/métodos , Acidaminococcus/enzimologia , Acidaminococcus/genética , Células HEK293 , Humanos
7.
Science ; 353(6299): aaf5573, 2016 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-27256883

RESUMO

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.


Assuntos
Imunidade Adaptativa/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Endorribonucleases/metabolismo , Leptotrichia/enzimologia , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endorribonucleases/química , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/virologia , Técnicas de Silenciamento de Genes , Loci Gênicos , Leptotrichia/genética , Leptotrichia/imunologia , Levivirus/imunologia , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Clivagem do RNA
8.
Sci Rep ; 5: 10833, 2015 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-26035283

RESUMO

Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients.


Assuntos
Sistemas CRISPR-Cas , DNA Viral/genética , DNA Viral/metabolismo , Vírus da Hepatite B/genética , Replicação Viral/genética , Animais , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA Circular , Modelos Animais de Doenças , Regulação Viral da Expressão Gênica , Marcação de Genes , Hepatite B/virologia , Humanos , Camundongos , RNA Guia de Cinetoplastídeos
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