13 research outputs found
High-Fidelity Identification of Single Nucleotide Polymorphism by Type V CRISPR Systems
Accurate and sensitive detection
of single nucleotide
polymorphism
(SNP) holds significant clinical implications, especially in the field
of cancer diagnosis. Leveraging its high accuracy and programmability,
the CRISPR system emerges as a promising platform for advancing the
identification of SNPs. In this study, we compared two type V CRISPR/Cas
systems (Cas12a and Cas14a) for the identification of cancer-related
SNP. Their identification performances were evaluated by characterizing
their mismatch tolerance to the BRAF gene. We found that the CRISPR/Cas14a
system exhibited superior accuracy and robustness over the CRISPR/Cas12a
system for SNP detection. Furthermore, blocker displacement amplification
(BDA) was combined with the CRISPR/Cas14a system to eliminate the
interference of the wild type (WT) and increase the detection accuracy.
In this strategy, we were able to detect BRAF V600E as low as 103 copies with a sensitivity of 0.1% variant allele frequency.
Moreover, the BDA-assisted CRISPR/Cas14a system has been applied to
identify the BRAF mutation from human colorectal carcinoma cells,
achieving a high sensitivity of 0.5% variant allele frequency, which
is comparable to or even superior to those of most commercially available
products. This work has broadened the scope of the CRISPR system
and provided a promising method for precision medicine
Novel Anti-CRISPR-Assisted CRISPR Biosensor for Exclusive Detection of Single-Stranded DNA (ssDNA)
Nucleic acid analysis
plays an important role in disease
diagnosis
and treatment. The discovery of CRISPR technology has provided novel
and versatile approaches to the detection of nucleic acids. However,
the most widely used CRISPR-Cas12a detection platforms lack the capability
to distinguish single-stranded DNA (ssDNA) from double-stranded DNA
(dsDNA). To overcome this limitation, we first employed an anti-CRISPR
protein (AcrVA1) to develop a novel CRISPR biosensor to detect ssDNA
exclusively. In this sensing strategy, AcrVA1 cut CRISPR guide RNA
(crRNA) to inhibit the cleavage activity of the CRISPR-Cas12a system.
Only ssDNA has the ability to recruit the cleaved crRNA fragment to
recover the detection ability of the CRISPR-Cas12 biosensor, but dsDNA
cannot accomplish this. By measuring the recovered cleavage activity
of the CRISPR-Cas12a biosensor, our developed AcrVA1-assisted CRISPR
biosensor is capable of distinguishing ssDNA from dsDNA, providing
a simple and reliable method for the detection of ssDNA. Furthermore,
we demonstrated our developed AcrVA1-assisted CRISPR biosensor to
monitor the enzymatic activity of helicase and screen its inhibitors
Electrochemical Detection of <i>Escherichia coli</i> from Aqueous Samples Using Engineered Phages
In
this study, an enzyme-based electrochemical method was developed
for the detection of <i>Escherichia coli</i> (<i>E.
coli</i>) using the T7 bacteriophages engineered with <i>lac</i>Z operon encoding for beta-galactosidase (β-gal).
The T7<sub><i>lac</i>Z</sub> phages can infect <i>E.
coli</i>, and have the ability to trigger the overexpression
of β-gal during the infection of <i>E. coli</i>. The
use of the engineered phages resulted in a more sensitive detection
of <i>E. coli</i> by (1) overexpression of β-gal in <i>E. coli</i> during the specific infection and (2) release of
the endogenous intracellular β-gal from <i>E. coli</i> following infection. The endogenous and phage-induced β-gal
was detected using the electrochemical method with 4-aminophenyl-β-galactopyranoside
(PAPG) as a substrate. The β-gal catalyzed PAPG to an electroactive
species <i>p</i>-aminophenol (PAP) which could be monitored
on an electrode. The electrochemical signal was proportional to the
concentration of <i>E. coli</i> in the original sample.
We demonstrated the application of our strategy in aqueous samples
(drinking water, apple juice, and skim milk). Using this method, we
were able to detect <i>E. coli</i> at the concentration
of approximately 10<sup>5</sup> CFU/mL in these aqueous samples in
3 h and 10<sup>2</sup> CFU/mL after 7 h. This strategy has the potential
to be extended to detect different bacteria using specific bacteriophages
engineered with gene encoding for appropriate enzymes
Lyophilized Engineered Phages for Escherichia coli Detection in Food Matrices
Ease of use, low
cost, and convenient transport are the key requirements
for a commercial bacteria detection kit designed for resource-limited
settings. Here, we report the colorimetric detection of Escherichia coli (E. coli) in food samples using freeze-dried engineered bacteriophages (phages).
In this approach, we have engineered T7 phages to carry the <i>lacZ</i> operon driven by T7 promoter to overexpress reporter
enzymes. The engineered phages were freeze-dried in a water-soluble
polymer for storage and transportation. When used for the detection
of E. coli cells, the intracellular
enzyme [β-galactosidase (β-gal)] was overexpressed and
released into the surrounding media, providing an enzyme-amplified
colorimetric signal. Using this strategy, we were able to detect E. coli cells at the concentration of 10<sup>2</sup> CFU mL<sup>–1</sup> in food samples without the need for
sophisticated instruments or skilled operators
Phage Predation Promotes Filamentous Bacterium <i>Piscinibacter</i> Colonization and Improves Structural and Hydraulic Stability of Microbial Aggregates
Although bacteria–phage interactions have broad
environmental
applications and ecological implications, the influence of phage predation
on bacterial aggregation and structural stability remains largely
unexplored. Herein, we demonstrate that inefficient lytic phage predation
can promote host filamentous bacterium Piscinibacter colonization onto non-host Thauera aggregates,
improving the structural and hydraulic stability of the dual-species
aggregates. Specifically, phage predation at 103–104 PFU/mL (i.e., multiplication of infection at 0.01–0.1)
promoted initial Piscinibacter colonization by 10–15
folds and resulted in 29–31% higher abundance of Piscinibacter in the stabilized aggregates than that in the control aggregates
without phage predation. Transcriptomic analysis revealed upregulated
genes related to quorum sensing (by 15–92 folds) and polysaccharide
secretion (by 10–90 folds) within the treated aggregates, which
was consistent with 120–172% higher content of polysaccharides
for the treated dual-species aggregates. Confocal laser scanning microscopic
images further confirmed the increase of filamentous bacteria and
polysaccharides (both with wider distribution) within the dual-species
aggregates. Accordlingly, the aggregates’ structural strength
(via atomic force microscopes) and shear resistance (via hydraulic
stress tests) increased by 77 and 42%, respectively, relative to the
control group. In the long-term experiments, the enhanced hydraulic
stability of the treated aggregates could facilitate dwelling bacteria
propagation in flow-through conditions. Overall, our study demonstrates
that phage predation can promote bacterial aggregation and enhance
aggregate structural stability, revealing the beneficial role of lytic
phage predation on bacterial symbiosis and environmental adaptivity
Image1_The lncRNA-mediated ceRNA network of Altica viridicyanea is involved in the regulation of the Toll/Imd signaling pathway under antibiotic treatment.TIF
Long noncoding RNAs (lncRNAs) play significant roles in the regulation of mRNA expression or in shaping the competing endogenous RNA (ceRNA) network by targeting miRNA. The insect gut is one of the most important tissues due to direct contact with external pathogens and functions in the immune defense against pathogen infection through the innate immune system and symbionts, but there are limited observations on the role of the lncRNA-involved ceRNA network of the Toll/Imd pathway and correlation analysis between this network and bacterial microbiota in the Altica viridicyanea gut. In this research, we constructed and sequenced six RNA sequencing libraries using normal and antibiotic-reared samples, generating a total of 17,193 lncRNAs and 26,361 mRNAs from massive clean data by quality control and bioinformatic analysis. Furthermore, a set of 8,539 differentially expressed lncRNAs (DELs) and 13,263 differentially expressed mRNAs (DEMs), of which related to various immune signaling pathways, such as the Toll/Imd, JAK/STAT, NF-κB, and PI3K-Akt signaling pathways, were obtained between the two experimental groups in A. viridicyanea. In addition, numerous GO and KEGG enrichment analyses were used to annotate the DELs and their target genes. Moreover, six Toll family members and nineteen signal genes from the Toll/Imd signaling pathway were identified and characterized using online tools, and phylogenetic analyses of the above genes proved their classification. Next, a lncRNA-miRNA-mRNA network of the Toll/Imd pathway was built, and it contained different numbers of DEMs in this pathway and related DELs based on prediction and annotation. In addition, qRT-PCR validation and sequencing data were conducted to show the expression patterns of the above DELs and DEMs related to the Toll/Imd signaling pathway. Finally, the correlated investigations between DELs or DEMs of the Toll/Imd signaling pathway and most changes in the gut bacterial microbiota revealed significantly positive or negative relationships between them. The present findings provide essential evidence for innate immune ceRNAs in the beetle gut and uncover new potential relationships between innate immune pathways and the gut bacterial microbiota in insects.</p
Table1_The lncRNA-mediated ceRNA network of Altica viridicyanea is involved in the regulation of the Toll/Imd signaling pathway under antibiotic treatment.XLSX
Long noncoding RNAs (lncRNAs) play significant roles in the regulation of mRNA expression or in shaping the competing endogenous RNA (ceRNA) network by targeting miRNA. The insect gut is one of the most important tissues due to direct contact with external pathogens and functions in the immune defense against pathogen infection through the innate immune system and symbionts, but there are limited observations on the role of the lncRNA-involved ceRNA network of the Toll/Imd pathway and correlation analysis between this network and bacterial microbiota in the Altica viridicyanea gut. In this research, we constructed and sequenced six RNA sequencing libraries using normal and antibiotic-reared samples, generating a total of 17,193 lncRNAs and 26,361 mRNAs from massive clean data by quality control and bioinformatic analysis. Furthermore, a set of 8,539 differentially expressed lncRNAs (DELs) and 13,263 differentially expressed mRNAs (DEMs), of which related to various immune signaling pathways, such as the Toll/Imd, JAK/STAT, NF-κB, and PI3K-Akt signaling pathways, were obtained between the two experimental groups in A. viridicyanea. In addition, numerous GO and KEGG enrichment analyses were used to annotate the DELs and their target genes. Moreover, six Toll family members and nineteen signal genes from the Toll/Imd signaling pathway were identified and characterized using online tools, and phylogenetic analyses of the above genes proved their classification. Next, a lncRNA-miRNA-mRNA network of the Toll/Imd pathway was built, and it contained different numbers of DEMs in this pathway and related DELs based on prediction and annotation. In addition, qRT-PCR validation and sequencing data were conducted to show the expression patterns of the above DELs and DEMs related to the Toll/Imd signaling pathway. Finally, the correlated investigations between DELs or DEMs of the Toll/Imd signaling pathway and most changes in the gut bacterial microbiota revealed significantly positive or negative relationships between them. The present findings provide essential evidence for innate immune ceRNAs in the beetle gut and uncover new potential relationships between innate immune pathways and the gut bacterial microbiota in insects.</p
Table3_The lncRNA-mediated ceRNA network of Altica viridicyanea is involved in the regulation of the Toll/Imd signaling pathway under antibiotic treatment.XLSX
Long noncoding RNAs (lncRNAs) play significant roles in the regulation of mRNA expression or in shaping the competing endogenous RNA (ceRNA) network by targeting miRNA. The insect gut is one of the most important tissues due to direct contact with external pathogens and functions in the immune defense against pathogen infection through the innate immune system and symbionts, but there are limited observations on the role of the lncRNA-involved ceRNA network of the Toll/Imd pathway and correlation analysis between this network and bacterial microbiota in the Altica viridicyanea gut. In this research, we constructed and sequenced six RNA sequencing libraries using normal and antibiotic-reared samples, generating a total of 17,193 lncRNAs and 26,361 mRNAs from massive clean data by quality control and bioinformatic analysis. Furthermore, a set of 8,539 differentially expressed lncRNAs (DELs) and 13,263 differentially expressed mRNAs (DEMs), of which related to various immune signaling pathways, such as the Toll/Imd, JAK/STAT, NF-κB, and PI3K-Akt signaling pathways, were obtained between the two experimental groups in A. viridicyanea. In addition, numerous GO and KEGG enrichment analyses were used to annotate the DELs and their target genes. Moreover, six Toll family members and nineteen signal genes from the Toll/Imd signaling pathway were identified and characterized using online tools, and phylogenetic analyses of the above genes proved their classification. Next, a lncRNA-miRNA-mRNA network of the Toll/Imd pathway was built, and it contained different numbers of DEMs in this pathway and related DELs based on prediction and annotation. In addition, qRT-PCR validation and sequencing data were conducted to show the expression patterns of the above DELs and DEMs related to the Toll/Imd signaling pathway. Finally, the correlated investigations between DELs or DEMs of the Toll/Imd signaling pathway and most changes in the gut bacterial microbiota revealed significantly positive or negative relationships between them. The present findings provide essential evidence for innate immune ceRNAs in the beetle gut and uncover new potential relationships between innate immune pathways and the gut bacterial microbiota in insects.</p
Table2_The lncRNA-mediated ceRNA network of Altica viridicyanea is involved in the regulation of the Toll/Imd signaling pathway under antibiotic treatment.XLSX
Long noncoding RNAs (lncRNAs) play significant roles in the regulation of mRNA expression or in shaping the competing endogenous RNA (ceRNA) network by targeting miRNA. The insect gut is one of the most important tissues due to direct contact with external pathogens and functions in the immune defense against pathogen infection through the innate immune system and symbionts, but there are limited observations on the role of the lncRNA-involved ceRNA network of the Toll/Imd pathway and correlation analysis between this network and bacterial microbiota in the Altica viridicyanea gut. In this research, we constructed and sequenced six RNA sequencing libraries using normal and antibiotic-reared samples, generating a total of 17,193 lncRNAs and 26,361 mRNAs from massive clean data by quality control and bioinformatic analysis. Furthermore, a set of 8,539 differentially expressed lncRNAs (DELs) and 13,263 differentially expressed mRNAs (DEMs), of which related to various immune signaling pathways, such as the Toll/Imd, JAK/STAT, NF-κB, and PI3K-Akt signaling pathways, were obtained between the two experimental groups in A. viridicyanea. In addition, numerous GO and KEGG enrichment analyses were used to annotate the DELs and their target genes. Moreover, six Toll family members and nineteen signal genes from the Toll/Imd signaling pathway were identified and characterized using online tools, and phylogenetic analyses of the above genes proved their classification. Next, a lncRNA-miRNA-mRNA network of the Toll/Imd pathway was built, and it contained different numbers of DEMs in this pathway and related DELs based on prediction and annotation. In addition, qRT-PCR validation and sequencing data were conducted to show the expression patterns of the above DELs and DEMs related to the Toll/Imd signaling pathway. Finally, the correlated investigations between DELs or DEMs of the Toll/Imd signaling pathway and most changes in the gut bacterial microbiota revealed significantly positive or negative relationships between them. The present findings provide essential evidence for innate immune ceRNAs in the beetle gut and uncover new potential relationships between innate immune pathways and the gut bacterial microbiota in insects.</p
Image2_The lncRNA-mediated ceRNA network of Altica viridicyanea is involved in the regulation of the Toll/Imd signaling pathway under antibiotic treatment.TIF
Long noncoding RNAs (lncRNAs) play significant roles in the regulation of mRNA expression or in shaping the competing endogenous RNA (ceRNA) network by targeting miRNA. The insect gut is one of the most important tissues due to direct contact with external pathogens and functions in the immune defense against pathogen infection through the innate immune system and symbionts, but there are limited observations on the role of the lncRNA-involved ceRNA network of the Toll/Imd pathway and correlation analysis between this network and bacterial microbiota in the Altica viridicyanea gut. In this research, we constructed and sequenced six RNA sequencing libraries using normal and antibiotic-reared samples, generating a total of 17,193 lncRNAs and 26,361 mRNAs from massive clean data by quality control and bioinformatic analysis. Furthermore, a set of 8,539 differentially expressed lncRNAs (DELs) and 13,263 differentially expressed mRNAs (DEMs), of which related to various immune signaling pathways, such as the Toll/Imd, JAK/STAT, NF-κB, and PI3K-Akt signaling pathways, were obtained between the two experimental groups in A. viridicyanea. In addition, numerous GO and KEGG enrichment analyses were used to annotate the DELs and their target genes. Moreover, six Toll family members and nineteen signal genes from the Toll/Imd signaling pathway were identified and characterized using online tools, and phylogenetic analyses of the above genes proved their classification. Next, a lncRNA-miRNA-mRNA network of the Toll/Imd pathway was built, and it contained different numbers of DEMs in this pathway and related DELs based on prediction and annotation. In addition, qRT-PCR validation and sequencing data were conducted to show the expression patterns of the above DELs and DEMs related to the Toll/Imd signaling pathway. Finally, the correlated investigations between DELs or DEMs of the Toll/Imd signaling pathway and most changes in the gut bacterial microbiota revealed significantly positive or negative relationships between them. The present findings provide essential evidence for innate immune ceRNAs in the beetle gut and uncover new potential relationships between innate immune pathways and the gut bacterial microbiota in insects.</p