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2.
Nat Commun ; 10(1): 4284, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537810

RESUMO

Highly specific Cas9 nucleases derived from SpCas9 are valuable tools for genome editing, but their wide applications are hampered by a lack of knowledge governing guide RNA (gRNA) activity. Here, we perform a genome-scale screen to measure gRNA activity for two highly specific SpCas9 variants (eSpCas9(1.1) and SpCas9-HF1) and wild-type SpCas9 (WT-SpCas9) in human cells, and obtain indel rates of over 50,000 gRNAs for each nuclease, covering ~20,000 genes. We evaluate the contribution of 1,031 features to gRNA activity and develope models for activity prediction. Our data reveals that a combination of RNN with important biological features outperforms other models for activity prediction. We further demonstrate that our model outperforms other popular gRNA design tools. Finally, we develop an online design tool DeepHF for the three Cas9 nucleases. The database, as well as the designer tool, is freely accessible via a web server, http://www.DeepHF.com/ .


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Aprendizado Profundo , Edição de Genes/métodos , RNA Guia/genética , Animais , Sequência de Bases/genética , Linhagem Celular Tumoral , Endonucleases/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutação INDEL/genética , Internet , Camundongos , Regiões Promotoras Genéticas/genética
3.
Nucleic Acids Res ; 47(19): 10202-10211, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31504832

RESUMO

The association of reverse transcriptases (RTs) with CRISPR-Cas system has recently attracted interest because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA molecules. However, our understanding of this spacer acquisition process remains limited. We characterized the in vivo acquisition of spacers mediated by an RT-Cas1 fusion protein linked to a type III-D system from Vibrio vulnificus strain YJ016, and showed that the adaptation module, consisting of the RT-Cas1 fusion, two different Cas2 proteins (A and B) and one of the two CRISPR arrays, was completely functional in a heterologous host. We found that mutations of the active site of the RT domain significantly decreased the acquisition of new spacers and showed that this RT-Cas1-associated adaptation module was able to incorporate spacers from RNA molecules into the CRISPR array. We demonstrated that the two Cas2 proteins of the adaptation module were required for spacer acquisition. Furthermore, we found that several sequence-specific features were required for the acquisition and integration of spacers derived from any region of the genome, with no bias along the 5'and 3'ends of coding sequences. This study provides new insight into the RT-Cas1 fusion protein-mediated acquisition of spacers from RNA molecules.


Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endodesoxirribonucleases/genética , Genoma Bacteriano/genética , Plasmídeos/genética , RNA/genética , DNA Polimerase Dirigida por RNA , Vibrio vulnificus/genética
4.
Nat Commun ; 10(1): 3693, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451697

RESUMO

Transcriptional regulation by nuclease-deficient CRISPR/Cas is a popular and valuable tool for routine control of gene expression. CRISPR interference in bacteria can be reliably achieved with high efficiencies. Yet, options for CRISPR activation (CRISPRa) remained limited in flexibility and activity because they relied on σ70 promoters. Here we report a eukaryote-like bacterial CRISPRa system based on σ54-dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate σ54-dependent promoters with biotechnology relevance in non-model bacteria. It also supports orthogonal gene regulation on multiple levels. Combining our CRISPRa with dxCas9 further expands flexibility in DNA targeting, and boosts dynamic ranges into regimes that enable construction of cascaded CRISPRa circuits. Application-wise, we construct a reusable scanning platform for readily optimizing metabolic pathways without library reconstructions. This eukaryote-like CRISPRa system is therefore a powerful and versatile synthetic biology tool for diverse research and industrial applications.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Engenharia de Proteínas/métodos , RNA Polimerase Sigma 54/genética , Escherichia coli/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Redes e Vias Metabólicas/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , RNA Guia/genética , Ativação Transcricional/genética
5.
Nat Commun ; 10(1): 3728, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427601

RESUMO

Discovery of CRISPR-Cas systems is one of paramount importance in the field of microbiology. Currently, how CRISPR-Cas systems are finely regulated remains to be defined. Here we use small regulatory RNA (sRNA) library to screen sRNAs targeting type I-F CRISPR-Cas system through proximity ligation by T4 RNA ligase and find 34 sRNAs linking to CRISPR loci. Among 34 sRNAs for potential regulators of CRISPR, sRNA pant463 and PhrS enhance CRISPR loci transcription, while pant391 represses their transcription. We identify PhrS as a regulator of CRISPR-Cas by binding CRISPR leaders to suppress Rho-dependent transcription termination. PhrS-mediated anti-termination facilitates CRISPR locus transcription to generate CRISPR RNA (crRNA) and subsequently promotes CRISPR-Cas adaptive immunity against bacteriophage invasion. Furthermore, this also exists in type I-C/-E CRISPR-Cas, suggesting general regulatory mechanisms in bacteria kingdom. Our findings identify sRNAs as important regulators of CRISPR-Cas, extending roles of sRNAs in controlling bacterial physiology by promoting CRISPR-Cas adaptation priming.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Escherichia coli/genética , Pseudomonas aeruginosa/genética , RNA Bacteriano/biossíntese , Pequeno RNA não Traduzido/genética , Fator Rho/antagonistas & inibidores , Terminação da Transcrição Genética/fisiologia , Bacteriófagos/genética , Sistemas CRISPR-Cas/genética , Ensaios de Triagem em Larga Escala , RNA Bacteriano/genética
6.
Nucleic Acids Res ; 47(17): 9259-9270, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31392987

RESUMO

The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.


Assuntos
Nucleotídeos de Adenina/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Mycobacterium tuberculosis/genética , Oligorribonucleotídeos/genética , Imunidade Adaptativa/imunologia , Nucleotídeos de Adenina/biossíntese , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , Sequências Repetitivas Dispersas/genética , Sequências Repetitivas Dispersas/imunologia , Mycobacterium tuberculosis/imunologia , Oligorribonucleotídeos/biossíntese , Células Procarióticas/imunologia , Clivagem do RNA/genética , Clivagem do RNA/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia
7.
J Basic Microbiol ; 59(9): 901-913, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31347199

RESUMO

The CRISPR-Cas (clustered regular interspaced short palindromic repeats and CRISPR-associated proteins) system is a newly discovered immune defense system in the genome of prokaryotes, which can resist the invasion of foreign genetic elements, such as plasmids or phage. In this study, 154 strains of Staphylococcus published in the CRISPRDatabase and 171 strains included in NCBI were downloaded, the confirmed and questionable CRISPR loci of which were analyzed by bioinformatics methods, including their distribution, characteristics of the structure (including the direct repeats, spacers and cas genes), and the relationship between the presence of CRISPR and the mecA gene. Meanwhile, a comprehensive analysis of orphan CRISPR arrays was performed on this basis. A total of 196 confirmed and 1757 questionable CRISPR loci were found in 325 Staphylococcus genomes. Only 25 strains contained cas genes, which were classified into III-A (48.1%) and II-C (51.9%). The difference between the presence of the cas gene and the carrying rate of mecA was statistically significant, and they were negatively correlated. A total of 137 confirmed and 1755 questionable CRISPR loci were assumed to be false-CRISPR. The present study also analyzed the questionable CRISPR array for the first time while analyzing the confirmed CRISPR array in the Staphylococcal genome and screened the false-CRISPR elements in the orphan CRISPR array.


Assuntos
Sistemas CRISPR-Cas/genética , Genoma Bacteriano/genética , Staphylococcus/genética , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biologia Computacional , Bases de Dados Genéticas , Resistência a Meticilina/genética , Filogenia , Staphylococcus/classificação
8.
Nat Commun ; 10(1): 3144, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31316073

RESUMO

Capitalizing on the inherent multiplexing capability of AsCpf1, we developed a multiplexed, high-throughput screening strategy that minimizes library size without sacrificing gene targeting efficiency. We demonstrated that AsCpf1 can be used for functional genomics screenings and that an AsCpf1-based multiplexed library performs similarly as compared to currently available monocistronic CRISPR/Cas9 libraries, with only one vector required for each gene. We construct the smallest whole-genome CRISPR knock-out library, Mini-human, for the human genome (n = 17,032 constructs targeting 16,977 protein-coding genes), which performs favorably compared to conventional Cas9 libraries.


Assuntos
Sistemas CRISPR-Cas/genética , Biblioteca Gênica , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Humanos , RNA Guia/química
9.
Nat Commun ; 10(1): 3001, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278272

RESUMO

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.


Assuntos
Imunidade Adaptativa/genética , Sistemas CRISPR-Cas/genética , Staphylococcus epidermidis/genética , Thermus thermophilus/genética , Elongação da Transcrição Genética/imunologia , Bacteriófagos/imunologia , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Plasmídeos/imunologia , RNA Guia/genética , RNA Guia/imunologia , RNA Guia/metabolismo , Staphylococcus epidermidis/imunologia , Thermus thermophilus/imunologia
10.
Res Microbiol ; 170(6-7): 296-299, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31279087

RESUMO

Quorum sensing (QS) is a molecular communication system that bacteria use to harmonize the regulation of genes in a cell density-dependent manner. In proteobacteria, QS is involved, among others, in virulence, biofilm formation or CRISPR-Cas gene regulation. Here, we report for the first time the effect of a QS-interfering enzyme to alter the regulation of CRISPR-Cas systems in model and clinical strains of Pseudomonas aeruginosa, as well as in the marine bacterium Chromobacterium violaceum CV12472. The expression of CRISPR-Cas genes decreased in most cases suggesting that enzymatic disruption of QS is promising for modulating phage-bacteria interactions.


Assuntos
Acil-Butirolactonas/metabolismo , Sistemas CRISPR-Cas/genética , Chromobacterium/genética , Regulação Bacteriana da Expressão Gênica/genética , Pseudomonas aeruginosa/genética , Percepção de Quorum/genética , Bacteriófagos/genética , Bacteriófagos/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Chromobacterium/isolamento & purificação , Chromobacterium/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Humanos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo
11.
Nat Commun ; 10(1): 2948, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270316

RESUMO

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biblioteca Gênica , Técnicas Genéticas , RNA/biossíntese , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , DNA/genética , Endonucleases/metabolismo , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
12.
BMC Genomics ; 20(1): 567, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31288753

RESUMO

BACKGROUND: Sequencing of microbiomes has accelerated the characterization of the diversity of CRISPR-Cas immune systems. However, the utilization of next generation short read sequences for the characterization of CRISPR-Cas dynamics remains limited due to the repetitive nature of CRISPR arrays. CRISPR arrays are comprised of short spacer segments (derived from invaders' genomes) interspaced between flanking repeat sequences. The repetitive structure of CRISPR arrays poses a computational challenge for the accurate assembly of CRISPR arrays from short reads. In this paper we evaluate the use of long read sequences for the analysis of CRISPR-Cas system dynamics in microbiomes. RESULTS: We analyzed a dataset of Illumina's TruSeq Synthetic Long-Reads (SLR) derived from a gut microbiome. We showed that long reads captured CRISPR spacers at a high degree of redundancy, which highlights the spacer conservation of spacer sharing CRISPR variants, enabling the study of CRISPR array dynamics in ways difficult to achieve though short read sequences. We introduce compressed spacer graphs, a visual abstraction of spacer sharing CRISPR arrays, to provide a simplified view of complex organizational structures present within CRISPR array dynamics. Utilizing compressed spacer graphs, several key defining characteristics of CRISPR-Cas system dynamics were observed including spacer acquisition and loss events, conservation of the trailer end spacers, and CRISPR arrays' directionality (transcription orientation). Other result highlights include the observation of intense array contraction and expansion events, and reconstruction of a full-length genome for a potential invader (Faecalibacterium phage) based on identified spacers. CONCLUSION: We demonstrate in an in silico system that long reads provide the necessary context for characterizing the organization of CRISPR arrays in a microbiome, and reveal dynamic and evolutionary features of CRISPR-Cas systems in a microbial population.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Variação Genética , Microbiota/genética , DNA Intergênico/genética
13.
Life Sci ; 231: 116531, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31175856

RESUMO

BACKGROUND: The Proteus is one of the most common human and animal pathogens. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR/Cas) are inheritable genetic elements found in a variety of archaea and bacteria in the evolution, providing immune function against foreign invasion. OBJECTIVES: To analyze the characteristics and functions of the CRISPR/Cas system in Proteus genomes, as well as the internal and external factors affecting the system. METHODS: CRISPR loci were identified and divided into groups based on the repeat sequence in 96 Proteus strains by identification. Compared the RNA secondary structure and minimum free energy of CRISPR loci through bioinformatics, the evolution of cas genes, and the effects of related elements were also discussed. RESULTS: 85 CRISPR loci were identified and divided into six groups based on the sequence of repeats, and the more stable the secondary structure of RNA, the smaller the minimum free energy, the fewer base mutations in the repeat, the more stable the CRISPR and the more complete the evolution of the system. In addition, Cas1 gene can be a symbol to distinguish species to some extent. Of all the influencing factors, CRISPR/Cas had the greatest impact on plasmids. CONCLUSIONS: This study examined the diversity of CRISPR/Cas system in Proteus and found statistically significant positive/negative correlations between variety factors (the RNA stability, free energy, etc.) and the CRISPR locus, which played a vital role in regulating the CRISPR/Cas system.


Assuntos
Proteus/genética , Proteus/metabolismo , Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Biologia Computacional/métodos , Bases de Dados Genéticas , Plasmídeos/genética , RNA/genética
14.
Nucleic Acids Res ; 47(14): 7518-7531, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31219587

RESUMO

Acquiring foreign spacer DNA into the CRISPR locus is an essential primary step of the CRISPR-Cas pathway in prokaryotes for developing host immunity to mobile genetic elements. Here, we investigate spacer integration in vitro using proteins from Pyrococcus furiosus and demonstrate that Cas1 and Cas2 are sufficient to accurately integrate spacers into a minimal CRISPR locus. Using high-throughput sequencing, we identified high frequency spacer integration occurring at the same CRISPR repeat border sites utilized in vivo, as well as at several non-CRISPR plasmid sequences which share features with repeats. Analysis of non-CRISPR integration sites revealed that Cas1 and Cas2 are directed to catalyze full-site spacer integration at specific DNA stretches where guanines and/or cytosines are 30 base pairs apart and the intervening sequence harbors several positionally conserved bases. Moreover, assaying a series of CRISPR repeat mutations, followed by sequencing of the integration products, revealed that the specificity of integration is primarily directed by sequences at the leader-repeat junction as well as an adenine-rich sequence block in the mid-repeat. Together, our results indicate that P. furiosus Cas1 and Cas2 recognize multiple sequence features distributed over a 30 base pair DNA region for accurate spacer integration at the CRISPR repeat.


Assuntos
Proteínas Arqueais/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Endonucleases/genética , Pyrococcus furiosus/genética , Proteínas Arqueais/metabolismo , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , Endonucleases/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Plasmídeos/genética , Pyrococcus furiosus/metabolismo
15.
Insect Biochem Mol Biol ; 111: 103172, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31103783

RESUMO

Recent advancements in genetic engineering technology have led to the development of CRISPR interference (CRISPRi) as a precise tool for regulating gene expression. When CRISPR/dCas9 is fused with transcriptional repressors, the system can robustly silence endogenous gene expression. The CRISPR/Cas9 tool is a promising alternative in organisms (e.g., Bombyx mori) that do not respond to traditional gene suppression techniques, such as RNA interference (RNAi). However, transcriptional repressors remain poorly categorized in multiple cell types and species, leading to difficulties in optimizing performance and efficiency. Here, we tested CRISPRi usability and efficiency in Bombyx mori cells (BmE). We fused dCas9 to five transcriptional repressors including KRAB, Hairy, SID, SRDX, and ERD. All five constructs were efficient in BmE cells. In a proof-of-concept experiment, we showed that CRISPRi acting on BmSoxE (a gene involved in cell proliferation) could generate similar phenotypes as RNAi gene suppression. Moreover, CRISPRi has fewer off-target effects. Through co-transfection of BmE cells with sgRNAs, we also demonstrated that dCas9 could simultaneously repress the expression of multiple genes. Furthermore, we identified sgRNA distance from transcriptional start site (TSS) and the dCas9: sgRNA ratio as the two limiting factors of CRISPRi efficiency. Our results demonstrated that CRISPR/dCas9 is a viable and rapid alternative for functional investigations of the B. mori genome and perhaps other Lepidoptera insects.


Assuntos
Bombyx/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Transcrição Genética , Animais , Linhagem Celular , Expressão Gênica , Inativação Gênica , Interferência de RNA , RNA Guia
16.
Nat Commun ; 10(1): 2113, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068592

RESUMO

Gene editing by CRISPR/Cas9 is commonly used to generate germline mutations or perform in vitro screens, but applicability for in vivo screening has so far been limited. Recently, it was shown that in Drosophila, Cas9 expression could be limited to a desired group of cells, allowing tissue-specific mutagenesis. Here, we thoroughly characterize tissue-specific (ts)CRISPR within the complex neuronal system of the Drosophila mushroom body. We report the generation of a library of gRNA-expressing plasmids and fly lines using optimized tools, which provides a valuable resource to the fly community. We demonstrate the application of our library in a large-scale in vivo screen, which reveals insights into developmental neuronal remodeling.


Assuntos
Animais Geneticamente Modificados/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Drosophila/genética , Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas/genética , Feminino , Masculino , Corpos Pedunculados/metabolismo , Mutagênese , Sistema Nervoso/crescimento & desenvolvimento , Plasticidade Neuronal/genética , Neurônios/fisiologia , Plasmídeos/genética , RNA Guia/genética
17.
Nat Commun ; 10(1): 2233, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110232

RESUMO

The recently developed single-cell CRISPR screening techniques, independently termed Perturb-Seq, CRISP-seq, or CROP-seq, combine pooled CRISPR screening with single-cell RNA-seq to investigate functional CRISPR screening in a single-cell granularity. Here, we present MUSIC, an integrated pipeline for model-based understanding of single-cell CRISPR screening data. Comprehensive tests applied to all the publicly available data revealed that MUSIC accurately quantifies and prioritizes the individual gene perturbation effect on cell phenotypes with tolerance for the substantial noise that exists in such data analysis. MUSIC facilitates the single-cell CRISPR screening from three perspectives, i.e., prioritizing the gene perturbation effect as an overall perturbation effect, in a functional topic-specific way, and quantifying the relationships between different perturbations. In summary, MUSIC provides an effective and applicable solution to elucidate perturbation function and biologic circuits by a model-based quantitative analysis of single-cell-based CRISPR screening data.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Modelos Genéticos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Estudos de Viabilidade , Perfilação da Expressão Gênica/métodos , Técnicas de Silenciamento de Genes , Humanos , Células Jurkat , Células K562 , RNA Guia/genética
18.
Mol Biol (Mosk) ; 53(2): 311-323, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31099781

RESUMO

The CRISPR/Cas9 nuclease system can effectively suppress the replication of the hepatitis B virus (HBV), while covalently closed circular DNA (cccDNA), a highly resistant form of the virus, persists in the nuclei of infected cells. The most common outcome of DNA double-strand breaks (DSBs) in cccDNA caused by CRISPR/Cas9 is double-strand break repair by nonhomologous end-joining, which results in insertion/deletion mutations. Modulation of the DNA double-strand break repair pathways by small molecules was shown to stimulate CRISPR/Cas9 activity and may potentially be utilized to enhance the elimination of HBV cccDNA. In this work, we used inhibitors of homologous (RI-1) and nonhomologous (NU7026) end-joining and their combination to stimulate antiviral activity of CRISPR/Cas9 on two cell models of HBV in vitro, i.e., the HepG2-1.1merHBV cells containing the HBV genome under the tet-on regulated cytomegalovirus promoter and the HepG2-1.5merHBV cells containing constitutive expression of HBV RNA under the wild-type promoter. The treatment of the cells with RI-1 or NU7026 after lentiviral transduction of CRISPR/Cas9 drops the levels of cccDNA compared to the DMSO-treated control. RI-1 and NU7026 resulted in 5.0-6.5 times more significant reduction in the HBV cccDNA level compared to the mock-control. In conclusion, the inhibition of both homologous and nonhomologous DNA double-strand break repair pathways increases the elimination of HBV cccDNA by CRISPR/Cas9 system in vitro, which may potentially be utilized as a therapeutic approach to treat chronic hepatitis B.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/efeitos dos fármacos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , DNA Viral/metabolismo , Vírus da Hepatite B , Sistemas CRISPR-Cas/genética , DNA Circular/genética , DNA Circular/metabolismo , DNA Viral/genética
19.
Mol Genet Genomics ; 294(5): 1095-1105, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31098740

RESUMO

CRISPR/Cas is an adaptive immune system found in prokaryotes, with the main function of protecting these cells from invasion and possible death by mobile genetic elements. Pseudomonas aeruginosa is considered a model for type I-F CRISPR/Cas system studies. However, its CRISPR loci characteristics have not yet been thoroughly described, and its function has not yet been fully unraveled. The aims of this study were to find the frequency of the system in Brazilian clinical isolates; to identify the loci sequence, its spacer diversity and its origins; as well as to propose a unified spacer library to aid in future structural studies of the CRISPR loci of P. aeruginosa. We investigated types I-F and I-E gene markers to establish CRISPR/Cas typing, and observed two strains harboring both systems simultaneously, a very rare feature. Through amplification and sequencing of CRISPR loci related to type I-F system, we describe polymorphisms in DRs and 350 spacers, of which 97 are new. The spacers that were identified had their possible organisms or proteins of origin identified. Spacer arrays were grouped in five different CRISPR patterns and the plasticity was inferred by rearrangements in spacer arrays. Here, we perform the first detailed and focused description of CRISPR/Cas elements in Brazilian clinical strains of P. aeruginosa. Our findings reflect active and highly diverse CRISPR loci, and we suggest that CRISPR/Cas may also pose as a transcriptional regulatory mechanism. The structural and diversity features described here can provide insights into the function of CRISPR/Cas in this pathogen and help guide the development of new therapeutic strategies.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Pseudomonas aeruginosa/genética , Brasil , Marcadores Genéticos/genética , Transcrição Genética/genética
20.
Mol Genet Genomics ; 294(5): 1263-1275, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31134321

RESUMO

Clostridium perfringens is an important pathogen of human and livestock infections, posing a threat to health. The horizontal gene transfer (HGT) of plasmids that carry toxin-related genes is involved in C. perfringens pathogenicity. The CRISPR/Cas system, which has been identified in a wide range of prokaryotes, provides acquired immunity against HGT. However, information about the CRISPR/Cas system in Clostridium perfringens is still limited. In this study, 111 C. perfringens strains with publicly available genomes were used to analyze the occurrence and diversity of CRISPR/Cas system and evaluate the potential of CRISPR-based genotyping in this multi-host pathogen. A total of 59 out of the 111 genomes harbored at least one confirmed CRISPR array. Four CRISPR/Cas system subtypes, including subtypes IB, IIA, IIC, and IIID systems, were identified in 32 strains. Subtype IB system was the most prevalent in this species, which was subdivided into four subgroups displaying subgroup specificity in terms of cas gene content, repeat sequence content, and PAM. We showed that the CRISPR spacer polymorphism can be used for evolutionary studies, and that it can provide discriminatory power for typing strains. Nevertheless, the application of this approach was largely limited to strains that contain the CRISPR/Cas system. Spacer origin analysis revealed that approximately one-fifth of spacers showed significant matches to plasmids and phages, thereby suggesting the implication of CRISPR/Cas systems in controlling HGT. Collectively, our results provide new insights into the diversity and evolution of CRISPR/Cas system in C. perfringens.


Assuntos
Sistemas CRISPR-Cas/genética , Clostridium perfringens/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Bacteriófagos/genética , Biologia Computacional/métodos , Transferência Genética Horizontal/genética , Genoma Bacteriano/genética , Filogenia , Plasmídeos/genética , Polimorfismo Genético/genética
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