RESUMO
CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π - π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.
Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos de DNA Transponíveis , Escherichia coli , Transferência Genética Horizontal , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.
Assuntos
DNA de Cadeia Simples , Edição de Genes , Rad51 Recombinase , Reparo de DNA por Recombinação , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Humanos , Edição de Genes/métodos , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Sistemas CRISPR-Cas , Células HEK293 , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Reparo do DNA por Junção de ExtremidadesRESUMO
Due to limitations in conventional disease vector control strategies including the rise of insecticide resistance in natural populations of mosquitoes, genetic control strategies using CRISPR gene drive systems have been under serious consideration. The identification of CRISPR target sites in mosquito populations is a key aspect for developing efficient genetic vector control strategies. While genome-wide Cas9 target sites have been explored in mosquitoes, a precise evaluation of target sites focused on coding sequence (CDS) is lacking. Additionally, target site polymorphisms have not been characterized for other nucleases such as Cas12a, which require a different DNA recognition site (PAM) and would expand the accessibility of mosquito genomes for genetic engineering. We undertook a comprehensive analysis of potential target sites for both Cas9 and Cas12a nucleases within the genomes of natural populations of Anopheles gambiae and Aedes aegypti from multiple continents. We demonstrate that using two nucleases increases the number of targets per gene. Also, we identified differences in nucleotide diversity between North American and African Aedes populations, impacting the abundance of good target sites with a minimal degree of polymorphisms that can affect the binding of gRNA. Lastly, we screened for gRNAs targeting sex-determination genes that could be widely applicable for developing field genetic control strategies. Overall, this work highlights the utility of employing both Cas9 and Cas12a nucleases and underscores the importance of designing universal genetic strategies adaptable to diverse mosquito populations.
Assuntos
Aedes , Anopheles , Sistemas CRISPR-Cas , Animais , Anopheles/genética , Aedes/genética , Variação Genética , RNA Guia de Sistemas CRISPR-Cas/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Genoma de Inseto , Mosquitos Vetores/genética , Edição de Genes , Proteínas de BactériasRESUMO
Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage-all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , DNA/metabolismo , DNA/genética , RNA/metabolismo , RNA/genética , Clivagem do DNA , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Edição de Genes/métodos , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Francisella/genéticaRESUMO
R-loop-triggered collateral single-stranded DNA (ssDNA) nuclease activity within Class 1 Type I CRISPR-Cas systems holds immense potential for nucleic acid detection. However, the hyperactive ssDNase activity of Cas3 introduces unwanted noise and false-positive results. In this study, we identified a novel Type I-A Cas3 variant derived from Thermococcus siculi, which remains in an auto-inhibited state until it is triggered by Cascade complex and R-loop formation. This Type I-A CRISPR-Cas3 system not only exhibits an expanded protospacer adjacent motif (PAM) recognition capability but also demonstrates remarkable intolerance towards mismatched sequences. Furthermore, it exhibits dual activation modes-responding to both DNA and RNA targets. The culmination of our research efforts has led to the development of the Hyper-Active-Verification Establishment (HAVE, ). This innovation enables swift and precise human papillomavirus (HPV) diagnosis in clinical samples, providing a robust molecular diagnostic tool based on the Type I-A CRISPR-Cas3 system. Our findings contribute to understanding type I-A CRISPR-Cas3 system regulation and facilitate the creation of advanced diagnostic solutions with broad clinical applicability.
Assuntos
Sistemas CRISPR-Cas , Infecções por Papillomavirus , Humanos , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/genética , Papillomaviridae/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Papillomavirus HumanoRESUMO
A new study characterizes and improves a novel small Cas12a variant before adapting it for in vivo genome editing by delivery via adeno-associated virus (AAV) vectors, showcasing the potential of small CRISPR systems and their compatibility with viral vectors.
Assuntos
Sistemas CRISPR-Cas , Dependovirus , Edição de Genes , Vetores Genéticos , Edição de Genes/métodos , Dependovirus/genética , Sistemas CRISPR-Cas/genética , Vetores Genéticos/genética , Animais , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismoRESUMO
Avian leukosis viruses (ALVs) include a group of avian retroviruses primarily associated with neoplastic diseases in poultry, commonly referred to as avian leukosis. Belonging to different subgroups based on their envelope properties, ALV subgroups A, B, and J (ALV-A, ALV-B, and ALV-J) are the most widespread in poultry populations. Early identification and removal of virus-shedding birds from infected flocks are essential for the ALVs' eradication. Therefore, the development of rapid, accurate, simple-to-use, and cost effective on-site diagnostic methods for the detection of ALV subgroups is very important. Cas13a, an RNA-guided RNA endonuclease that cleaves target single-stranded RNA, also exhibits non-specific endonuclease activity on any bystander RNA in close proximity. The distinct trans-cleavage activity of Cas13 has been exploited in the molecular diagnosis of multiple pathogens including several viruses. Here, we describe the development and application of a highly sensitive Cas13a-based molecular test for the specific detection of proviral DNA of ALV-A, B, and J subgroups. Prokaryotically expressed LwaCas13a, purified through ion exchange and size-exclusion chromatography, was combined with recombinase polymerase amplification (RPA) and T7 transcription to establish the SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) molecular detection system for the detection of proviral DNA of ALV-A/B/J subgroups. This novel method that needs less sample input with a short turnaround time is based on isothermal detection at 37 °C with a color-based lateral flow readout. The detection limit of the assay for ALV-A/B/J subgroups was 50 copies with no cross reactivity with ALV-C/D/E subgroups and other avian oncogenic viruses such as reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). The development and evaluation of a highly sensitive and specific visual method of detection of ALV-A/B/J nucleic acids using CRISPR-Cas13a described here will help in ALV detection in eradication programs.
Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Sistemas CRISPR-Cas , DNA Viral , Provírus , Vírus da Leucose Aviária/genética , Vírus da Leucose Aviária/isolamento & purificação , Vírus da Leucose Aviária/classificação , Animais , Provírus/genética , Provírus/isolamento & purificação , Leucose Aviária/virologia , Leucose Aviária/diagnóstico , DNA Viral/genética , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/diagnóstico , Galinhas/virologia , Sensibilidade e Especificidade , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismoRESUMO
The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.
Assuntos
Acidaminococcus , Proteínas de Bactérias , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Domínio Catalítico , Microscopia Crioeletrônica , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Acidaminococcus/enzimologia , Acidaminococcus/genética , Acidaminococcus/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Estruturas R-Loop/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/química , RNA Guia de Sistemas CRISPR-Cas/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , Modelos Moleculares , Domínios Proteicos , Relação Estrutura-Atividade , Ligação ProteicaRESUMO
TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life. IS605-family TnpB homologs function as programmable RNA-guided homing endonucleases in bacteria, driving transposon maintenance through DNA double-strand break-stimulated homologous recombination. In this work, we uncovered molecular mechanisms of the transposition life cycle of IS607-family elements that, notably, also encode group I introns. We identified specific features for a candidate "IStron" from Clostridium botulinum that allow the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts evolved an ability to balance competing and mutually exclusive activities that promote selfish transposon spread while limiting adverse fitness costs on the host. Collectively, this work highlights molecular innovation in the multifunctional utility of transposon-encoded noncoding RNAs.
Assuntos
Proteínas de Bactérias , Proteínas Associadas a CRISPR , Clostridium botulinum , Elementos de DNA Transponíveis , Endodesoxirribonucleases , Íntrons , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas , Recombinação Homóloga , Splicing de RNA , RNA Guia de Sistemas CRISPR-Cas/genética , Transposases/metabolismo , Transposases/genética , Clostridium botulinum/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismoRESUMO
In the type III CRISPR system, cyclic oligoadenylate (cOA) molecules act as second messengers, activating various promiscuous ancillary nucleases that indiscriminately degrade host and viral DNA/RNA. Conversely, ring nucleases, by specifically cleaving cOA molecules, function as off-switches to protect host cells from dormancy or death, and allow viruses to counteract immune responses. The fusion protein Csx1-Crn2, combining host ribonuclease with viral ring nuclease, represents a unique self-limiting ribonuclease family. Here, we describe the structures of Csx1-Crn2 from the organism of Marinitoga sp., in both its full-length and truncated forms, as well as in complex with cA4. We show that Csx1-Crn2 operates as a homo-tetramer, a configuration crucial for preserving the structural integrity of the HEPN domain and ensuring effective ssRNA cleavage. The binding of cA4 to the CARF domain triggers significant conformational changes across the CARF, HTH, and into the HEPN domains, leading the two R-X4-6-H motifs to form a composite catalytic site. Intriguingly, an acetate ion was found to bind at this composite site by mimicking the scissile phosphate. Further molecular docking analysis reveals that the HEPN domain can accommodate a single ssRNA molecule involving both R-X4-6-H motifs, underscoring the importance of HEPN domain dimerization for its activation.
Assuntos
Proteínas de Bactérias , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Ribonucleases/metabolismo , Ribonucleases/química , Ribonucleases/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Modelos Moleculares , Ligação Proteica , Nucleotídeos de Adenina/metabolismo , Nucleotídeos de Adenina/química , Multimerização Proteica , Domínios Proteicos , OligorribonucleotídeosRESUMO
Precise gene regulation and programmable RNA editing are vital RNA-level regulatory mechanisms. Gene repression tools grounded in small non-coding RNAs, microRNAs, and CRISPR-dCas proteins, along with RNA editing tools anchored in Adenosine Deaminases acting on RNA (ADARs), have found extensive application in molecular biology and cellular engineering. Here, we introduced a novel approach wherein we developed an EcCas6e mediated crRNA-mRNA annealing system for gene repression in Escherichia coli and RNA editing in Saccharomyces cerevisiae. We found that EcCas6e possesses inherent RNA annealing ability attributed to a secondary positively charged cleft, enhancing crRNA-mRNA hybridization and stability. Based on this, we demonstrated that EcCas6e, along with its cognate crRNA repeat containing a complementary region to the ribosome binding site of a target mRNA, effectively represses gene expression up to 25-fold. Furthermore, we demonstrated that multiple crRNAs can be easily assembled and can simultaneously target up to 13 genes. Lastly, the EcCas6e-crRNA system was developed as an RNA editing tool by fusing it with the ADAR2 deaminase domain. The EcCas6e-crRNA mediated gene repression and RNA editing tools hold broad applications for research and biotechnology.
Assuntos
Escherichia coli , Edição de RNA , RNA Antissenso , RNA Mensageiro , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adenosina Desaminase/metabolismo , Adenosina Desaminase/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Sistemas CRISPR-Cas , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genéticaRESUMO
Point of care testing (POCT) of nucleic acid (NA) contributes to the timely disease diagnosis, like bacteria and virus screening in households or resource-constrained areas, but its development has always been stagnant. Herein, we proposed an exonuclease III cascaded with CRISPR/Cas12a (Exo-III/Cas12a) amplification strategy and constructed a smartphone-based portable fluorescence detector (SPFD) to repurpose the commercial alpha-fetoprotein (AFP) strip for the ultrasensitive and hand-held detection of NA samples. In detail, the target-initiated-Exo-III/Cas12a strategy realizes the signal amplification and liberates AFP from magnetic beads through the trans-cleavages of activated Cas12a toward the AFP aptamer. After magnetic separation and migration, the fluorescence signals of the test (FT) and control (FC) lines on the AFP strip were digitally output by the SPFD, and the FT/FC was employed for the quantitative analysis to minimize external disturbances and improve accuracy. We experimentally assessed the universe applicability of the proposed NA-POCT platform toward miRNA-155, 16S rRNA of Staphylococcus aureus, and ORF1a/b RNA of Covid-19 pseudovirus, achieving favorable detection limits of 42 aM, 18 CFU/mL, and 87 copies/µL, respectively. Moreover, its simplicity, universality, and admirable detection performance demonstrate a great potential in the aspect of rapidly transforming the existing POCT devices for multiple new applications at the time of need.
Assuntos
Exodesoxirribonucleases , Técnicas de Amplificação de Ácido Nucleico , Testes Imediatos , Smartphone , alfa-Fetoproteínas , alfa-Fetoproteínas/análise , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/química , Humanos , Sistemas CRISPR-Cas , Fluorescência , Proteínas Associadas a CRISPR/metabolismo , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , Limite de Detecção , Técnicas Biossensoriais/métodos , RNA Viral/análise , Espectrometria de Fluorescência , Proteínas de Bactérias , EndodesoxirribonucleasesRESUMO
In biological systems, the activities of macromolecular complexes must sometimes be turned off. Thus, a wide variety of protein inhibitors has evolved for this purpose. These inhibitors function through diverse mechanisms, including steric blocking of crucial interactions, enzymatic modification of key residues or substrates, and perturbation of post-translational modifications1. Anti-CRISPRs-proteins that block the activity of CRISPR-Cas systems-are one of the largest groups of inhibitors described, with more than 90 families that function through diverse mechanisms2-4. Here, we characterize the anti-CRISPR AcrIF25, and we show that it inhibits the type I-F CRISPR-Cas system by pulling apart the fully assembled effector complex. AcrIF25 binds to the predominant CRISPR RNA-binding components of this complex, comprising six Cas7 subunits, and strips them from the RNA. Structural and biochemical studies indicate that AcrIF25 removes one Cas7 subunit at a time, starting at one end of the complex. Notably, this feat is achieved with no apparent enzymatic activity. To our knowledge, AcrIF25 is the first example of a protein that disassembles a large and stable macromolecular complex in the absence of an external energy source. As such, AcrIF25 establishes a paradigm for macromolecular complex inhibitors that may be used for biotechnological applications.
Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Substâncias Macromoleculares , Proteínas Virais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/química , Modelos Moleculares , Ligação Proteica , Subunidades Proteicas/metabolismo , Subunidades Proteicas/química , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Biotecnologia/tendências , Bacteriófagos , Proteínas Virais/metabolismoRESUMO
The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a padlock activator which could inhibit the trans-cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca2+ sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca2+ dependent. In the presence of Ca2+, DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the trans-cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca2+ concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg2+, Mn2+, Cd2+, Cu2+, Na+, Al3+, K+, Fe2+, and Fe3+ indicated high selectivity. Recovery ranged from 93.32% to 103.28% with RSDs from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca2+ in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system.
Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , Cálcio , Técnicas Biossensoriais/métodos , Cálcio/metabolismo , Cálcio/química , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , DNA Catalítico/química , Animais , Leite/química , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Limite de DetecçãoRESUMO
The healthcare burden imposed by bacterial infections demands robust and accessible diagnostic methods that can be performed outside hospitals and centralized laboratories. Here, we report Pathogen Assay with Ratiometric Luminescence (PEARL), a sensitive and easy-to-operate platform for detecting pathogenic bacteria. The PEARL leveraged a color-changeable CRISPR-Cas12a sensor and recombinase polymerase amplification to elicit ratiometric bioluminescence responses to target inputs. This platform enabled robust and visualized identification of attomolar bacteria genome deoxyribonucleic acid according to the color changes of the reactions. In addition, the components of the color-changeable Cas12a sensor could be lyophilized for 3 month storage at ambient temperature and then be fully activated with the amplicons derived from crude bacterial lysates, reducing the requirements for cold-chain storage and tedious handling steps. We demonstrated that the PEARL assay is applicable for identifying the infections caused by Pseudomonas aeruginosa in different clinical specimens, including sputa, urines, and swabs derived from wounds. These results revealed the potential of PEARL to be used by untrained personnel, which will facilitate decentralized pathogen diagnosis in community- and resource-limited regions.
Assuntos
Sistemas CRISPR-Cas , Medições Luminescentes , Pseudomonas aeruginosa , Sistemas CRISPR-Cas/genética , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/genética , Humanos , Liofilização , Cor , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Técnicas Biossensoriais , Proteínas Associadas a CRISPR/metabolismo , LuminescênciaRESUMO
Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.
Assuntos
Arabidopsis , Marcação de Genes , Arabidopsis/genética , Marcação de Genes/métodos , Transformação Genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Plantas Geneticamente Modificadas/genéticaRESUMO
DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a trans-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the trans-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 U mL-1. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.
Assuntos
Sistemas CRISPR-Cas , Uracila-DNA Glicosidase , Uracila-DNA Glicosidase/metabolismo , Uracila-DNA Glicosidase/genética , Sistemas CRISPR-Cas/genética , Humanos , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genéticaRESUMO
In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.
Assuntos
Bacteriófagos , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA , Regulação Viral da Expressão Gênica , Sequências Hélice-Volta-Hélice , Proteínas de Ligação a RNA , Proteínas Virais , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/ultraestrutura , Sítios de Ligação , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas Associadas a CRISPR/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Genes Virais , Modelos Moleculares , Conformação de Ácido Nucleico , Pectobacterium carotovorum/virologia , Biossíntese de Proteínas/genética , Domínios Proteicos , Ribossomos/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/ultraestrutura , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , RNA Viral/ultraestrutura , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Especificidade por Substrato , Transcrição Gênica , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/ultraestruturaRESUMO
The diagnosis of dengue virus (DENV) has been challenging particularly in areas far from clinical laboratories. Early diagnosis of pathogens is a prerequisite for the timely treatment and pathogen control. An ideal diagnostic for viral infections should possess high sensitivity, specificity, and flexibility. In this study, we implemented dual amplification involving Cas13a and Cas12a, enabling sensitive and visually aided diagnostics for the dengue virus. Cas13a recognized the target RNA by crRNA and formed the assembly of the Cas13a/crRNA/RNA ternary complex, engaged in collateral cleavage of nearby crRNA of Cas12a. The Cas12a/crRNA/dsDNA activator ternary complex could not be assembled due to the absence of crRNA of Cas12a. Moreover, the probe, with 5' and 3' termini labeled with FAM and biotin, could not be separated. The probes labeled with FAM and biotin, combined the Anti-FAM and the Anti-Biotin Ab-coated gold nanoparticle, and conformed sandwich structure on the T-line. The red line on the paper strip caused by clumping of AuNPs on the T-line indicated the detection of dengue virus. This technique, utilizing an activated Cas13a system cleaving the crRNA of Cas12a, triggered a cascade that amplifies the virus signal, achieving a low detection limit of 190 fM with fluorescence. Moreover, even at 1 pM, the red color on the T-line was easily visible by naked eyes. The developed strategy, incorporating cascade enzymatic amplification, exhibited good sensitivity and may serve as a field-deployable diagnostic tool for dengue virus.
Assuntos
Vírus da Dengue , Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise , Proteínas Associadas a CRISPR/metabolismo , Nanopartículas Metálicas/química , Limite de Detecção , Ouro/química , Proteínas de Bactérias , EndodesoxirribonucleasesRESUMO
The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3'-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.