scholarly journals Transcriptional Knockdown in Pneumococci Using CRISPR Interference

2019 ◽  
pp. 89-98
Author(s):  
Morten Kjos
Keyword(s):  
Crispr Interference

scholarly journals A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Nicole A. Ellis ◽  
Byoungkwan Kim ◽  
Jessica Tung ◽  
Matthias P. Machner
Keyword(s):  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Polymerase Activity ◽  
Growth Defect ◽  
Great Promise ◽  
Macrophage Infection ◽  
Crispr Interference ◽  
Crispr Array ◽  
Virulence Protein

AbstractCatalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

CRISPR interference and its applications

2021 ◽  
Author(s):  
Seyedkhashayar Ghavami ◽  
Amir Pandi
Keyword(s):  
Crispr Interference

scholarly journals Impact of CRISPR interference on strain development in biotechnology

2020 ◽  
Vol 67 (1) ◽  
pp. 7-21 ◽  
Author(s):  
Kerstin Schultenkämper ◽  
Luciana F. Brito ◽  
Volker F. Wendisch
Keyword(s):  
Strain Development ◽  
Crispr Interference

scholarly journals LncRNA FAM83H-AS1 Amplification is Associated With a Poor Prognosis in Lung Adenocarcinoma and Can Serve as A Therapeutic Target

2020 ◽  
Author(s):  
Siwei Wang ◽  
Chencheng Han ◽  
Tongyan Liu ◽  
Zhifei Ma ◽  
Mantang Qiu ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Poor Prognosis ◽  
Therapeutic Potential ◽  
Gene Annotation ◽  
Tumor Xenograft ◽  
Driver Gene ◽  
Clinical Prognosis ◽  
Crispr Interference ◽  
Oncogenic Driver

Abstract Background: Few oncogenic drivers of long noncoding RNAs (lncRNAs) have been identified and investigated. Identifying noncoding drivers provides potential strategies for novel interventions in lung adenocarcinoma (LUAD). Methods: We constructed a machine learning model for driver gene annotation using pan-cancer and clinical prognosis data from OncoKB and TCGA to predict potential oncogenic drivers of lncRNAs; then, we used zebrafish models to validate the biological function of candidate targets. The full length of FAM83H-AS1 was obtained by rapid amplification of the cDNA ends (RACE) assay. RNA pull-down, RNA immunoprecipitation (RIP), quantative mass spectrometry (QMS) and RNA sequencing (RNA-Seq) assays were utilized to explore the potential mechanisms. Additionally, we used CRISPR interference (CRISPRi) system and patient-derived tumor xenograft (PDTX) model to evaluate the therapeutic potential of targeting FAM83H-AS1 in vivo.Results: The results suggested that FAM83H-AS1 was a potential oncogenic driver from the chromosome 8q24 amplicon; increases in the expression of FAM83H-AS1 resulted in poor prognosis for LUAD patients both in JSCH and TCGA cohorts. Functional assays revealed that FAM83H-AS1 promotes malignant progression and inhibits apoptosis. Mechanistically, FAM83H-AS1 binds with HNRNPK to enhance the translation of oncogenes RAB8B and RAB14. Experiments using CRISPR interference (CRISPRi)-mediated xenografts and patient-derived tumor xenograft (PDTX) models indicated that targeting FAM83H-AS1 inhibited LUAD progression in vivo. Conclusions: Our work demonstrated that FAM83H-AS1 is a potential oncogenic driver that inhibits LUAD-mediated apoptosis via the FAM83H-AS1-HNRNPK-RAB8B/RAB14 axis. Importantly, we suggest targeting of FAM83H-AS1 as a potential therapeutic strategy for LUAD.

scholarly journals Regulation of Microbial Metabolic Rates Using CRISPR Interference With Expanded PAM Sequences

2020 ◽  
Vol 11 ◽  
Author(s):  
Bumjoon Kim ◽  
Hyun Ju Kim ◽  
Sang Jun Lee
Keyword(s):  
Metabolic Rates ◽  
Crispr Interference

Tailoring bacterial cellulose structure through CRISPR interference‐mediated downregulation of galU in Komagataeibacter xylinus CGMCC 2955

2020 ◽  
Vol 117 (7) ◽  
pp. 2165-2176 ◽  
Author(s):  
Long‐Hui Huang ◽  
Qi‐Jing Liu ◽  
Xue‐Wen Sun ◽  
Xue‐Jing Li ◽  
Miao Liu ◽  
...  
Keyword(s):  
Bacterial Cellulose ◽  
Cellulose Structure ◽  
Crispr Interference ◽  
Komagataeibacter Xylinus

scholarly journals CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1843-1845 ◽  
Author(s):  
Luciano A. Marraffini ◽  
Erik J. Sontheimer
Keyword(s):  
Antibiotic Resistance ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Staphylococcus Epidermidis ◽  
Pathogenic Bacteria ◽  
Target Sequence ◽  
Crispr Interference ◽  
Multiple Routes ◽  
Short Palindromic Repeat ◽  
Self Splicing

Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate ofStaphylococcus epidermidisharbors a CRISPR spacer that matches thenickasegene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation inS. epidermidis. Insertion of a self-splicing intron intonickaseblocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.

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