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1.
Food Chem ; 334: 127608, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32711280

RESUMO

Food analysis to ensure food safety and quality are relevant to all countries. This study aimed to develop a detection technique by combining recombinase polymerase amplification with CRISPR-Cas12a for food safety (termed RPA-Cas12a-FS). Our data showed that this novel method could be detected via fluorescence intensity for the molecular identification of foodborne pathogenic bacteria, genetically modified crops, and meat adulteration. After optimization, the sensitivity and stability of RPA-Cas12a-FS was further enhanced. The RPA-Cas12a-FS system could specifically detect target gene levels as low as 10 copies in 45 min at 37 °C. The RPA-Cas12a-FS system was sensitive both using standard samples in the lab and using samples from the field, which indicated that this detection method was practical. In conclusion, a simple, rapid, and highly sensitive detection method based on CRISPR-Cas12a was developed for molecular identification in the food safety field without requiring technical expertise or ancillary equipment.


Assuntos
Sistemas CRISPR-Cas , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Microbiologia de Alimentos/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Produtos Agrícolas/genética , Endodesoxirribonucleases/genética , Fluorescência , Inocuidade dos Alimentos , Carne , Plantas Geneticamente Modificadas/genética , RNA Guia , Recombinases/genética , Sensibilidade e Especificidade
2.
PLoS Genet ; 16(9): e1009001, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886661

RESUMO

During meiosis, diploid organisms reduce their chromosome number by half to generate haploid gametes. This process depends on the repair of double strand DNA breaks as crossover recombination events between homologous chromosomes, which hold homologs together to ensure their proper segregation to opposite spindle poles during the first meiotic division. Although most organisms are limited in the number of crossovers between homologs by a phenomenon called crossover interference, the consequences of excess interfering crossovers on meiotic chromosome segregation are not well known. Here we show that extra interfering crossovers lead to a range of meiotic defects and we uncover mechanisms that counteract these errors. Using chromosomes that exhibit a high frequency of supernumerary crossovers in Caenorhabditis elegans, we find that essential chromosomal structures are mispatterned in the presence of multiple crossovers, subjecting chromosomes to improper spindle forces and leading to defects in metaphase alignment. Additionally, the chromosomes with extra interfering crossovers often exhibited segregation defects in anaphase I, with a high incidence of chromatin bridges that sometimes created a tether between the chromosome and the first polar body. However, these anaphase I bridges were often able to resolve in a LEM-3 nuclease dependent manner, and chromosome tethers that persisted were frequently resolved during Meiosis II by a second mechanism that preferentially segregates the tethered sister chromatid into the polar body. Altogether these findings demonstrate that excess interfering crossovers can severely impact chromosome patterning and segregation, highlighting the importance of limiting the number of recombination events between homologous chromosomes for the proper execution of meiosis.


Assuntos
Segregação de Cromossomos/genética , Troca Genética/genética , Meiose/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cromátides/genética , Cromatina/genética , Posicionamento Cromossômico/genética , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Recombinação Genética
3.
Nucleic Acids Res ; 48(17): e101, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32797156

RESUMO

Recent efforts in biological engineering have made detection of nucleic acids in samples more rapid, inexpensive and sensitive using CRISPR-based approaches. We expand one of these Cas13a-based methods to detect small molecules in a one-batch assay. Using SHERLOCK-based profiling of in vitrotranscription (SPRINT), in vitro transcribed RNA sequence-specifically triggers the RNase activity of Cas13a. This event activates its non-specific RNase activity, which enables cleavage of an RNA oligonucleotide labeled with a quencher/fluorophore pair and thereby de-quenches the fluorophore. This fluorogenic output can be measured to assess transcriptional output. The use of riboswitches or proteins to regulate transcription via specific effector molecules is leveraged as a coupled assay that transforms effector concentration into fluorescence intensity. In this way, we quantified eight different compounds, including cofactors, nucleotides, metabolites of amino acids, tetracycline and monatomic ions in samples. In this manner, hundreds of reactions can be easily quantified in a few hours. This increased throughput also enables detailed characterization of transcriptional regulators, synthetic compounds that inhibit transcription, or other coupled enzymatic reactions. These SPRINT reactions are easily adaptable to portable formats and could therefore be used for the detection of analytes in the field or at point-of-care situations.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/métodos , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/metabolismo , Ensaios Enzimáticos/métodos , Inibidores da Síntese de Ácido Nucleico/análise , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Corantes Fluorescentes/química , Leptotrichia , Ligantes , Inibidores da Síntese de Ácido Nucleico/farmacologia , Riboswitch , Rifampina/análise , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos dos fármacos
4.
Proc Natl Acad Sci U S A ; 117(37): 22890-22899, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32843348

RESUMO

CRISPR-Cas genome engineering has revolutionized biomedical research by enabling targeted genome modification with unprecedented ease. In the popular model organism Drosophila melanogaster, gene editing has so far relied exclusively on the prototypical CRISPR nuclease Cas9. Additional CRISPR systems could expand the genomic target space, offer additional modes of regulation, and enable the independent manipulation of genes in different cells of the same animal. Here we describe a platform for efficient Cas12a gene editing in Drosophila We show that Cas12a from Lachnospiraceae bacterium, but not Acidaminococcus spec., can mediate robust gene editing in vivo. In combination with most CRISPR RNAs (crRNAs), LbCas12a activity is high at 29 °C, but low at 18 °C, enabling modulation of gene editing by temperature. LbCas12a can directly utilize compact crRNA arrays that are substantially easier to construct than Cas9 single-guide RNA arrays, facilitating multiplex genome engineering. Furthermore, we show that conditional expression of LbCas12a is sufficient to mediate tightly controlled gene editing in a variety of tissues, allowing detailed analysis of gene function in a multicellular organism. We also test a variant of LbCas12a with a D156R point mutation and show that it has substantially higher activity and outperforms a state-of-the-art Cas9 system in identifying essential genes. Cas12a gene editing expands the genome-engineering toolbox in Drosophila and will be a powerful method for the functional annotation of the genome. This work also presents a fully genetically encoded Cas12a system in an animal, laying out principles for the development of similar systems in other genetically tractable organisms for multiplexed conditional genome engineering.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Drosophila melanogaster/genética , Endonucleases/metabolismo , RNA/genética , RNA/metabolismo , RNA Guia/metabolismo
5.
Nucleic Acids Res ; 48(14): 8165-8177, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32609820

RESUMO

In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nt guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging approximately nine generations, a dCas12a oscillator design with 40-nt CRISPR RNAs performed much better, having a strongly repressed off-state, distinct autocorrelation function peaks, and an average peak-to-peak period length of ∼7.5 generations. Along with free-running oscillator circuits, we measure repression response times in open-loop systems with inducible RNA steps to compare with oscillator period times. We track thousands of cells for 24+ h at the single-cell level using a microfluidic device. In creating a circuit with nearly translationally independent behavior, as the RNAs control each others' transcription, we present the possibility for a synthetic oscillator generalizable across many organisms and readily linkable for transcriptional control.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Microfluídica/métodos , Periodicidade , RNA Guia/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli , Microfluídica/instrumentação , RNA Guia/genética , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
6.
Nat Commun ; 11(1): 3455, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661245

RESUMO

CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 in leukemia cells.


Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Edição de Genes , Regulação Leucêmica da Expressão Gênica , Leucemia/genética , Animais , Proliferação de Células , Epigênese Genética , Biblioteca Gênica , Engenharia Genética , Genoma Humano , Células HEK293 , Humanos , Células K562 , Camundongos , Células NIH 3T3 , Domínios Proteicos , RNA Guia/genética
7.
J Biosci Bioeng ; 130(4): 367-373, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32646632

RESUMO

Cross hybridization breeding of sake yeasts is hampered by difficulty in acquisition of haploid cells through sporulation. We previously demonstrated that typical sake yeast strains were defective in meiotic chromosome recombination, which caused poor sporulation and loss of spore viability. In this study, we screened a single copy plasmid genomic DNA library of the laboratory Saccharomyces cerevisiae GRF88 for genes that might complement the meiotic recombination defect of UTCAH-3, a strain derived from the sake yeast Kyokai no. 7 (K7). We identified the SPO11 gene of the laboratory strain (ScSPO11), encoding a meiosis-specific endonuclease that catalyzes DNA double-strand breaks required for meiotic recombination, as a gene that restored meiotic recombination and spore viability of UTCAH-3. K7SPO11 could not restore sporulation efficiency and spore viability of UTCAH-3 and a laboratory strain BY4743 spo11Δ/spo11Δ, indicating that K7SPO11 is not functional. Sequence analysis of the SPO11 genes of various Kyokai sake yeasts (K1, and K3-K10) revealed that the K7 group of sake yeasts (K6, K7, K9, and K10) had a mutual missense mutation (C73T) in addition to other three common mutations present in all Kyokai yeasts tested. ScSPO11C73T created through in vitro mutagenesis could not restore spore viability of BY4743 spo11Δ/spo11Δ. On the other hand, K8SPO11, which have the three common mutations except for C73T could restore spore viability of BY4743 spo11Δ/spo11Δ. These results suggest that C73T might be a causative mutation of recombination defect in K7SPO11. Moreover, we found that the introduction of ScRIM15 restored sporulation efficiency but not spore viability.


Assuntos
Bebidas Alcoólicas/microbiologia , Endodesoxirribonucleases/genética , Meiose/genética , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Clonagem Molecular , Quebras de DNA de Cadeia Dupla , Mutação , Saccharomyces cerevisiae/citologia
8.
Mol Cell ; 79(3): 416-424.e5, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32645367

RESUMO

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Genoma Bacteriano/imunologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Bactérias/classificação , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico
9.
Proc Natl Acad Sci U S A ; 117(28): 16527-16536, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601218

RESUMO

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.


Assuntos
Endodesoxirribonucleases/metabolismo , Ácido Fólico/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Mitose , Rad51 Recombinase/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Humanos , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo
10.
Gene ; 757: 144929, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32622990

RESUMO

Phaeodactylum tricornutum is a model microalgae that is widely used to study diatom physiology and ecology. Since the meiotic process and sexual cycle have never been observed directly, P. tricornutum has been considered to be an asexual species. However, phylogenetic analysis of the P. tricornutum genome has revealed a series of meiosis-specific gene homologues in this species. We identified two copies of differently transcribed SPO11 homologs that contain the conserved motifs of Winged-helix and Toprim domains. The homolog PtSPO11-3 interacts with TopoVIB in yeast two-hybrid analysis, whereas the homolog PtSPO11-2 could rescue the sporulation defect of a Spo11 yeast mutant strain. PtSPO11-2 was also found to be significantly up-regulated at low temperatures in P. tricornutum and its key catalytic residue was important to the homolog's function in sporulation. The results herein provide positive clue that meiosis and sexual reproduction could exist in this diatom.


Assuntos
Domínio Catalítico , Diatomáceas/genética , Endodesoxirribonucleases/metabolismo , Meiose , Microalgas/genética , Sequência Conservada , Diatomáceas/fisiologia , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Microalgas/fisiologia , Ligação Proteica , Multimerização Proteica
11.
Nucleic Acids Res ; 48(15): 8474-8489, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32652040

RESUMO

Highly toxic DNA double-strand breaks (DSBs) readily trigger the DNA damage response (DDR) in cells, which delays cell cycle progression to ensure proper DSB repair. In Saccharomyces cerevisiae, mitotic S phase (20-30 min) is lengthened upon DNA damage. During meiosis, Spo11-induced DSB onset and repair lasts up to 5 h. We report that the NH2-terminal domain (NTD; residues 1-66) of Rad51 has dual functions for repairing DSBs during vegetative growth and meiosis. Firstly, Rad51-NTD exhibits autonomous expression-enhancing activity for high-level production of native Rad51 and when fused to exogenous ß-galactosidase in vivo. Secondly, Rad51-NTD is an S/T-Q cluster domain (SCD) harboring three putative Mec1/Tel1 target sites. Mec1/Tel1-dependent phosphorylation antagonizes the proteasomal degradation pathway, increasing the half-life of Rad51 from ∼30 min to ≥180 min. Our results evidence a direct link between homologous recombination and DDR modulated by Rad51 homeostasis.


Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Meiose/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/genética , Domínios Proteicos/genética , Proteínas Serina-Treonina Quinases/genética , Proteólise , Saccharomyces cerevisiae/genética , beta-Galactosidase/genética
12.
Nucleic Acids Res ; 48(15): 8601-8616, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32687187

RESUMO

The CRISPR-Cas9 system is widely used for target-specific genome engineering. CRISPR-Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.


Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA/genética , Endodesoxirribonucleases/genética , RNA Guia/genética , Pareamento Incorreto de Bases/genética , Clivagem do DNA , Edição de Genes , Humanos , Modelos Moleculares , Mutação/genética , Conformação de Ácido Nucleico , RNA/genética , RNA Circular/genética
13.
PLoS One ; 15(6): e0235218, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32584890

RESUMO

Previous research regarding Holstein cows has mainly focused on increasing milk yield. However, in order to maximize the economical profits of Holstein cattle farming, it is necessary to fully take advantage of Holstein bulls to produce high-grade beef. The present study aims to investigate different transcriptomic profiling of Holstein bulls and steers, via high-throughput RNA-sequencing (RNA-seq). The growth and beef quality traits of Holstein steers and bulls were characterized via assessment of weight, rib eye area, marbling score, shear force and intramuscular fat percentage of the longissimus lumborum (LL) muscle. The results indicated that castration improved the meat quality, yet reduced the meat yield. Subsequently, RNA-seq of the LL muscle from Holstein steers and bulls revealed a total of 56 differentially expressed genes (DEGs). We performed the functional enrichment analysis in Gene Ontology (GO) annotations of the DEGs using GOseq R package software and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using KOBAS tool. Through the integrated analysis of DEGs with reported QTLs and SNPs, seven promising candidate genes potentially affecting the beef quality of LL muscle following castration were discovered, including muscle structural protein coding genes (MYH1, MYH4, MYH10) and functional protein coding genes (GADL1, CYP2R1, EEPD1, SHISA3). Among them, MYH10, GADL1, CYP2R1, EEPD1 and SHISA3 were novel candidate genes associated with beef quality traits. Notably, EEPD1 was associated with both meat quality and reproduction traits, thus indicating its overlapping role in responding to hormone change, and subsequently inducing beef quality improvement. Our findings provide a complete dataset of gene expression profile of LL in Holstein bulls and steers, and will aid in understanding how castration influence meat yield and quality.


Assuntos
Qualidade dos Alimentos , Carne/análise , Músculo Esquelético/metabolismo , Transcriptoma , Animais , Carboxiliases/genética , Bovinos , Endodesoxirribonucleases/genética , Feminino , Masculino , Cadeias Pesadas de Miosina/genética , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , Análise de Sequência de RNA
14.
Proc Natl Acad Sci U S A ; 117(26): 14936-14947, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541055

RESUMO

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.


Assuntos
Proteínas Arqueais/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteína Homóloga a MRE11/metabolismo , Sulfolobus acidocaldarius/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , DNA Arqueal/química , DNA Arqueal/genética , DNA Arqueal/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Proteína Homóloga a MRE11/química , Proteína Homóloga a MRE11/genética , Microscopia de Força Atômica , Ligação Proteica , Sulfolobus acidocaldarius/química , Sulfolobus acidocaldarius/enzimologia , Sulfolobus acidocaldarius/metabolismo
15.
Am J Hum Genet ; 107(1): 96-110, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32589923

RESUMO

A recent genome-wide association study of Huntington disease (HD) implicated genes involved in DNA maintenance processes as modifiers of onset, including multiple genome-wide significant signals in a chr15 region containing the DNA repair gene Fanconi-Associated Nuclease 1 (FAN1). Here, we have carried out detailed genetic, molecular, and cellular investigation of the modifiers at this locus. We find that missense changes within or near the DNA-binding domain (p.Arg507His and p.Arg377Trp) reduce FAN1's DNA-binding activity and its capacity to rescue mitomycin C-induced cytotoxicity, accounting for two infrequent onset-hastening modifier signals. We also idenified a third onset-hastening modifier signal whose mechanism of action remains uncertain but does not involve an amino acid change in FAN1. We present additional evidence that a frequent onset-delaying modifier signal does not alter FAN1 coding sequence but is associated with increased FAN1 mRNA expression in the cerebral cortex. Consistent with these findings and other cellular overexpression and/or suppression studies, knockout of FAN1 increased CAG repeat expansion in HD-induced pluripotent stem cells. Together, these studies support the process of somatic CAG repeat expansion as a therapeutic target in HD, and they clearly indicate that multiple genetic variations act by different means through FAN1 to influence HD onset in a manner that is largely additive, except in the rare circumstance that two onset-hastening alleles are present. Thus, an individual's particular combination of FAN1 haplotypes may influence their suitability for HD clinical trials, particularly if the therapeutic agent aims to reduce CAG repeat instability.


Assuntos
Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Doença de Huntington/genética , Enzimas Multifuncionais/genética , Linhagem Celular , Estudo de Associação Genômica Ampla/métodos , Células HEK293 , Haplótipos/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética
16.
Nat Biotechnol ; 38(9): 1054-1060, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32393821

RESUMO

Only female insects transmit diseases such as malaria, dengue and Zika; therefore, control methods that bias the sex ratio of insect offspring have long been sought. Genetic elements such as sex-chromosome drives can distort sex ratios to produce unisex populations that eventually collapse, but the underlying molecular mechanisms are unknown. We report a male-biased sex-distorter gene drive (SDGD) in the human malaria vector Anopheles gambiae. We induced super-Mendelian inheritance of the X-chromosome-shredding I-PpoI nuclease by coupling this to a CRISPR-based gene drive inserted into a conserved sequence of the doublesex (dsx) gene. In modeling of invasion dynamics, SDGD was predicted to have a quicker impact on female mosquito populations than previously developed gene drives targeting female fertility. The SDGD at the dsx locus led to a male-only population from a 2.5% starting allelic frequency in 10-14 generations, with population collapse and no selection for resistance. Our results support the use of SDGD for malaria vector control.


Assuntos
Anopheles/genética , Tecnologia de Impulso Genético/métodos , Malária/transmissão , Mosquitos Vetores/genética , Processos de Determinação Sexual/genética , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Malária/prevenção & controle , Masculino , Controle de Mosquitos , Cromossomo X/genética , Cromossomo X/metabolismo
17.
PLoS Genet ; 16(5): e1008787, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32392243

RESUMO

During repair of DNA double-strand breaks, resection of DNA ends influences how these lesions will be repaired. If resection is activated, the break will be channeled through homologous recombination; if not, it will be simply ligated using the non-homologous end-joining machinery. Regulation of resection relies greatly on modulating CtIP, which can be done by modifying: i) its interaction partners, ii) its post-translational modifications, or iii) its cellular levels, by regulating transcription, splicing and/or protein stability/degradation. Here, we have analyzed the role of ALC1, a chromatin remodeler previously described as an integral part of the DNA damage response, in resection. Strikingly, we found that ALC1 affects resection independently of chromatin remodeling activity or its ability to bind damaged chromatin. In fact, it cooperates with the RNA-helicase eIF4A1 to help stabilize the most abundant splicing form of CtIP mRNA. This function relies on the presence of a specific RNA sequence in the 5' UTR of CtIP. Therefore, we describe an additional layer of regulation of CtIP-at the level of mRNA stability through ALC1 and eIF4A1.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Regiões 5' não Traduzidas , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Células HeLa , Recombinação Homóloga , Humanos , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Mensageiro/química
18.
Proc Natl Acad Sci U S A ; 117(21): 11274-11282, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32376630

RESUMO

The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has led to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable nonspecific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of nuclease-dead Cas12a (dCas12a) from Francisella novicida as it inspects and binds to its DNA target. Our results reveal concealed thermodynamic factors affecting dCas12a DNA binding, which should guide the design and optimization of crRNA that limits off-target effects, including the crucial role of an extended protospacer adjacent motif (PAM) sequence and the impact of the specific base composition of crRNA-DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endodesoxirribonucleases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Escherichia coli/genética , Francisella , Ensaios de Triagem em Larga Escala/métodos , Interferência de RNA , RNA Guia , Termodinâmica
19.
Am J Med Sci ; 360(3): 287-292, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32387117

RESUMO

Immunoglobulin A (IgA) nephropathy is one of the most common glomerulonephritis characterized by the deposition of IgA in glomerular mesangium. Karyomegalic interstitial nephritis (KIN) is a rare interstitial nephritis with potential hereditary factors. IgA nephropathy concomitant with KIN has not yet been reported. Herein, we describe the clinical course, ultrasonic images and gastrointestinal endoscopy findings of a 28-year-old-male patient with IgA nephropathy with KIN. The pathologic examination of the renal biopsy specimen demonstrated mild mesangial proliferative IgA nephropathy with KIN. Molecular genetic testing detected an abnormality in FAN1 gene. The heterozygous mutation was present on chromosome 15q13.3. However, IgA nephropathy with KIN is a rare disorder, and its pathogenesis is yet to be clarified.


Assuntos
Glomerulonefrite por IGA/patologia , Nefrite Intersticial/genética , Nefrite Intersticial/patologia , Adulto , Cromossomos Humanos Par 15/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Mutação da Fase de Leitura , Mesângio Glomerular , Glomerulonefrite por IGA/tratamento farmacológico , Glomerulonefrite por IGA/fisiopatologia , Heterozigoto , Humanos , Testes de Função Renal , Masculino , Enzimas Multifuncionais/genética , Mutação de Sentido Incorreto , Nefrite Intersticial/fisiopatologia
20.
Nat Commun ; 11(1): 2598, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451402

RESUMO

DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.


Assuntos
Mutação , Oócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reparo de DNA por Recombinação/genética , Aneuploidia , Animais , Apoptose , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Feminino , Fertilização , Genes bcl-2 , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Proteínas de Ligação a Fosfato/deficiência , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína X Associada a bcl-2/deficiência , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
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