Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1011465 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25859262.fastq.gz | Fastqsanger.gz |
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Project description:Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools. Here, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs (PAMs) based on bioinformatics analysis combined with plasmid interference assay in S. spinosa. By reprogramming this endogenous CRISPR-Cas system, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the Restriction-Modification (RM) systems was systematically disarmed. We showed that by disarming one type I RM system and two type II RM systems simultaneously, the transformation efficiency of foreign plasmids was significantly increased. Based on the engineered strain with simultaneous deletion of these three RM genes, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster as well as gene insertion at high efficiency. Collectively, we developed a reliable and high-efficiency genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.
2025-09-25 | GSE278034 | GEO
Project description:Reprogramming the endogenous type III-A CRISPR-Cas system for genome editing and RNA interference in Mycobacterium tuberculosis
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Project description:The CRISPR-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpyCas9) is most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system imposes limits on target selection and multiplexed genome engineering. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from Streptococcus pyogenes (SpyCas9), Streptococcus aureus (SauCas9), Lachnospiraceae bacterium (LbaCas12a, previously known as LbCpf1), Acidaminococcus sp. (AspCas12a, previously known as AsCpf1) and Neisseria meningitidis (Nme2Cas9) are orthogonal systems capable of operating simultaneously in zebrafish. We implemented multichannel CRISPR recording using up to three CRISPR systems, and show that LbaCas12a may provide superior information density compared to previous methods. We also demonstrate that type II Acrs (anti-CRISPRs) are effective inhibitors of SpyCas9 in zebrafish. These results indicate that at least five CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These orthogonal CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing and genetic recording in animals.
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Project description:Understanding constraints which shape antibiotic resistance is key for predicting and controlling drug resistance. Here, we performed high-throughput laboratory evolution of Actinobacillus pleuropneumoniae and its ciprofloxacin resistance-inducing derivatives.This study aims to explore the mechanism of acquired ciprofloxacin resistance in Actinobacillus pleuropneumoniae.
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Project description:To reveal the transcriptional profiles of Actinobacillus pleuropneumoniae under biofilm and planktonic growth, we established a biofilm-forming culture method and constructed a mutant strain Δpga with defect in biofilm formation. Wild-type and Δpga mutant strains of Actinobacillus pleuropneumoniae strain 4074 were cultured in bottles with shaking for planktonic (WT_PK) and in microplates in static status for biofilm (WT_BF, Δpga), respectively. The bacteria in logarithmic growth period of different culture groups were collected for RNA seq.
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Project description:Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows for robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.
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Project description:The type V-I CRISPR-Cas system is becoming increasingly attractive for its potential utility in gene editing. However, natural nucleases often exhibit low efficiency, limiting their application. Here, we utilized structure-guided rational design and combinatorial protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. Accordingly, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene-editing activity in mammalian cells and plants. Cas-SF01 displays comparable or superior editing performance compared to SpCas9 or recently engineered Cas12 nucleases. Further analysis of PAM recognition showed that Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. Additionally, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01HiFi (high-fidelity Cas-SF01). Finally, we demonstrated that Cas-SF01 has robust gene-editing activity in both the monocot plant rice and dicot plant pepper. Our results suggest that Cas-SF01 can serve as a robust gene-editing platform with high efficiency and specificity for future genome editing applications across different organisms.
2023-11-05 | GSE236755 | GEO
Project description:Simple and efficient delivery of CRISPR genome editing systems in primary cells remains a major challenge. Here, we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells. PAGE couples a cell-penetrating Cas protein with a cell-penetrating endosomal escape peptide in a 30-minute incubation that yields up to ~98% editing efficiency in primary human and mouse T cells. PAGE provides a broadly generalizable platform for next generation genome engineering in primary cells. CITATION INFORMATION: Zhang Zhen, Baxter Amy E, Ren Diqiu, Qin Kunhua, Chen Zeyu, Collins Sierra M., Huang Hua, Komar Chad A., Bailer Peter F., Parker Jared B., Blobel Gerd A., Kohli Rahul M., Wherry E. John*, Berger Shelley,*, and Shi Junwei*. Peptide-assisted genome editing permits efficient CRISPR engineering of primary T cells.
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Project description:Investigation of whole genome changes in six serotypes of Actinobacillus pleuropneumoniae in control cultures compared to bacteria grown in media containg the iron chelator 2,2'-dipyridyl.
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Project description:To determine the role of Actinobacillus pleuropneumoniae two-component system QseBQseC, we constructed a qseBqseC gene-deleted mutant ΔqseBΔqseC based on the wild type A. pleuropneumoniae 4074. The transcriptional profiles were compared between the A. pleuropneumoniae ΔqseBΔqseC and its parental strain under the normal growth condition using microarray. A total of 44 genes were found differentially expressed (DE) compared to the wild type strain. These functional genes are primarily related to metabolism, cell wall biogenesis, energy, replication and recombination. Further investigations indicated that the type IV pili (Tfp) assembly protein PilM is regulated directly by QseB, and PilM is essential for adherence and virulence. Characterization of the QseBQseC regulon genes will provides new insight into understanding of the relevant signal transduction pathways and prevention of the infection.
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