Efficient CRISPR-Mediated Post-Transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression.
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ABSTRACT:
SUBMITTER: Zebec Z
PROVIDER: S-EPMC5068938 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundCRISPR/Cas9 mediated gene knockout is a powerful tool for genome editing with the ability to target multiple genes simultaneously. Establishing an efficient, multiplexed gene knockout system using CRISPR/Cas9 that is both simple and robust in its application would further advance the adoption of CRISPR/Cas9 for genetic studies.ResultsIn this study, we present a simple, versatile and highly efficient method to achieve acute gene knockout with CRISPR/Cas9 using chemically synthesized crRNA and tracrRNA oligos. We demonstrate that co-transfection of the crRNA:tracrRNA duplex into Cas9-expressing cells leads to target gene mutation and loss of target protein expression in the majority of the cell population. We also show that delivering three crRNAs targeting EGFP, KRAS and PTEN in the same reaction leads to the simultaneous knockout of all three genes. Direct comparison of multiplexed gene targeting by crRNA:tracrRNA and by siRNA indicates that these two methods are comparable in their efficiency and kinetics of gene silencing.ConclusionsOur method is a convenient yet powerful tool to enable rapid and scalable gene knockout using CRISPR/Cas9 in mammalian cells.
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Project description:The existence of a global gene regulatory system in the hyperthermophilic archaeon Sulfolobus solfataricus is described. The system is responsive to carbon source quality and acts at the level of transcription to coordinate synthesis of three physically unlinked glycosyl hydrolases implicated in carbohydrate utilization. The specific activities of three enzymes, an alpha-glucosidase (malA), a beta-glycosidase (lacS), and an alpha-amylase, were reduced 4-, 20-, and 10-fold, respectively, in response to the addition of supplementary carbon sources to a minimal sucrose medium. Western blot analysis using anti-alpha-glucosidase and anti-beta-glycosidase antibodies indicated that reduced enzyme activities resulted exclusively from decreased enzyme levels. Northern blot analysis of malA and lacS mRNAs revealed that changes in enzyme abundance arose primarily from reductions in transcript concentrations. Culture conditions precipitating rapid changes in lacS gene expression were established to determine the response time of the regulatory system in vivo. Full induction occurred within a single generation whereas full repression occurred more slowly, requiring nearly 38 generations. Since lacS mRNA abundance changed much more rapidly in response to a nutrient down shift than to a nutrient up shift, transcript synthesis rather than degradation likely plays a role in the regulatory response.
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Project description:The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C).
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Project description:Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these alpha-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize maltodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-alpha-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of alpha-glucan substrates by P. furiosus.
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Project description:Hepatitis B virus (HBV) infection is a major risk factor of liver cirrhosis and hepatocellular carcinoma. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been used to precisely edit the HBV genome and eliminate HBV through non-homologous end-joining repair of double-stranded break (DSB). However, the CRISPR/Cas9-mediated DSB triggers instability of host genome and exhibits low efficiency to edit genome, limiting its application. CRISPR cytidine base editors (CBEs) could silence genes by generating a premature stop codon. Here we developed a CRISPR base editor approach to precisely edit single nucleotide within the HBV genome to impair HBV gene expression. Specifically, a single-guide RNA (sgRNA) was designed to edit the 30th codon of HBV S gene, which encodes HBV surface antigen (HBsAg), from CAG (glutamine) to stop codon TAG. We next used human hepatoma PLC/PRF/5 cells carrying the HBV genome to establish a cell line that expresses a CBE (PLC/PRF/5-CBE). Lentivirus was used to introduce sgRNA into PLC/PRF/5-CBE cells. Phenotypically, 71% of PLC/PRF/5-CBE cells developed a premature stop codon within the S gene. Levels of HBs messenger RNA were significantly decreased. A 92% reduction of HBsAg secretion was observed in PLC/PRF/5-CBE cells. The intracellular HBsAg was also reduced by 84% after treatment of gRNA_S. Furthermore, no off-target effect was detected in predicted off-target loci within the HBV genome. Sequencing confirmed that 95%, 93%, 93%, 9%, and 72% S gene sequences of HBV genotypes B, C, F, G, and H had the binding site of sgRNA. Conclusion: Our findings indicate that CRISPR-mediated base editing is an efficient approach to silence the HBV S gene, suggesting its therapeutic potential to eliminate HBV.
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Project description:Bacterial immune systems, such as CRISPR-Cas or restriction-modification (R-M) systems, affect bacterial pathogenicity and antibiotic resistance by modulating horizontal gene flow. A model system for CRISPR-Cas regulation, the Type I-E system from Escherichia coli, is silent under standard laboratory conditions and experimentally observing the dynamics of CRISPR-Cas activation is challenging. Two characteristic features of CRISPR-Cas regulation in E. coli are cooperative transcription repression of cas gene and CRISPR array promoters, and fast non-specific degradation of full length CRISPR transcripts (pre-crRNA). In this work, we use computational modeling to understand how these features affect the system expression dynamics. Signaling which leads to CRISPR-Cas activation is currently unknown, so to bypass this step, we here propose a conceptual setup for cas expression activation, where cas genes are put under transcription control typical for a restriction-modification (R-M) system and then introduced into a cell. Known transcription regulation of an R-M system is used as a proxy for currently unknown CRISPR-Cas transcription control, as both systems are characterized by high cooperativity, which is likely related to similar dynamical constraints of their function. We find that the two characteristic CRISPR-Cas control features are responsible for its temporally-specific dynamical response, so that the system makes a steep (switch-like) transition from OFF to ON state with a time-delay controlled by pre-crRNA degradation rate. We furthermore find that cooperative transcription regulation qualitatively leads to a cross-over to a regime where, at higher pre-crRNA processing rates, crRNA generation approaches the limit of an infinitely abrupt system induction. We propose that these dynamical properties are associated with rapid expression of CRISPR-Cas components and efficient protection of bacterial cells against foreign DNA. In terms of synthetic applications, the setup proposed here should allow highly efficient expression of small RNAs in a narrow time interval, with a specified time-delay with respect to the signal onset.
| S-EPMC5675862 | biostudies-literature
Project description:CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity.
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Project description:There is a growing body of evidence about the presence and the activity of the miRISC in the nucleus of mammalian cells. Here, we show by quantitative proteomic analysis that Ago2 interacts with the nucleoplasmic protein Sfpq in an RNA-dependent fashion. By a combination of HITS-CLIP and transcriptomic analyses, we demonstrate that Sfpq directly controls the miRNA targeting of a subset of binding sites by local binding. Sfpq modulates miRNA targeting in both nucleoplasm and cytoplasm, indicating a nucleoplasmic commitment of Sfpq-target mRNAs that globally influences miRNA modes of action. Mechanistically, Sfpq binds to a sizeable set of long 3'UTRs forming aggregates to optimize miRNA positioning/recruitment at selected binding sites, including let-7a binding to Lin28A 3'UTR. Our results extend the miRNA-mediated post-transcriptional gene silencing into the nucleoplasm and indicate that an Sfpq-dependent strategy for controlling miRNA activity takes place in cells, contributing to the complexity of miRNA-dependent gene expression control.
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Project description:Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing and transcriptional regulation. In this review, we discuss multiplexed CRISPR technologies and describe methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo. Applications that benefit from multiplexed CRISPR technologies, including cellular recorders, genetic circuits, biosensors, combinatorial genetic perturbations, large-scale genome engineering and the rewiring of metabolic pathways, are highlighted. We also offer a glimpse of emerging challenges and emphasize experimental considerations for future studies.
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Project description:The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-mediated virus defense represents an adaptive immune system in many bacteria and archaea. Small CRISPR RNAs cause cleavage of complementary invading nucleic acids in conjunction with an associated protein or a protein complex. Here, we show CRISPR-mediated cleavage of mRNA from an invading virus in the hyperthermophilic archaeon Sulfolobus solfataricus. More than 40% of the targeted mRNA could be cleaved, as demonstrated by quantitative polymerase chain reaction. Cleavage of the mRNA was visualized by northern analyses and cleavage sites were mapped. In vitro, the same substrates were cleaved by the purified CRISPR-associated CMR complex from Sulfolobus solfataricus. The in vivo system was also re-programmed to knock down mRNA of a selected chromosomal gene (?-galactosidase) using an artificial miniCRISPR locus. With a single complementary spacer, ?50% reduction of the targeted mRNA and of corresponding intracellular protein activity was achieved. Our results demonstrate in vivo cleavage of mRNA in a prokaryote mediated by small RNAs (i.e. analogous to RNA interference in eukaryotes) and the re-programming of the system to silence specific genes of interest.
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