Dataset Information

Effective Blocking of Microbial Transcriptional Initiation by dCas9-NG-Mediated CRISPR Interference.

ABSTRACT: CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence has been constructed by removing the dependence on the last base G of PAM through protein engineering of Cas9. In this study, a dCas9-NG protein was engineered by introducing two active site mutations in Cas9-NG, and its ability to regulate transcription was evaluated in the gal promoter in E. coli. Analysis of cell growth rate, D-galactose consumption rate, and gal transcripts confirmed that dCas9-NG can completely repress the promoter by recognizing DNA targets with PAM of 5'-NGG, NGA, NGC, NGT, and NAG. Our study showed possible PAM sequences for dCas9-NG and provided information on target-specific sgRNA design for regulation of both gene expression and cellular metabolism.

SUBMITTER: Kim B

PROVIDER: S-EPMC9728369 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Effective Blocking of Microbial Transcriptional Initiation by dCas9-NG-Mediated CRISPR Interference.

Kim Bumjoon B Kim Hyun Ju HJ Lee Sang Jun SJ

Journal of microbiology and biotechnology 20201201 12

CRISPR interference (CRISPRi) has been developed as a transcriptional control tool by inactivating the DNA cleavage ability of Cas9 nucleases to produce dCas9 (deactivated Cas9), and leaving dCas9 the ability to specifically bind to the target DNA sequence. CRISPR/Cas9 technology has limitations in designing target-specific single-guide RNA (sgRNA) due to the dependence of protospacer adjacent motif (PAM) (5'-NGG) for binding target DNAs. Reportedly, Cas9-NG recognizing 5'-NG as the PAM sequence ...[more]

PMID: 32958732

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Project description:Magnaporthe oryzae and Ustilaginoidea virens are two filamentous fungal pathogens that threaten rice production worldwide. Genetic tools that permit fast gene deletion and silencing are of great interest for functional genomics of fungal pathogens. As a revolutionary genome editing tool, clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) enable many innovative applications. Here, we developed a CRISPR interference (CRISPRi) toolkit using nuclease activity dead Cas9 (dCas9) to silence genes of interest in M. oryzae and U. virens. We optimized the components of CRISPRi vectors, including transcriptional repression domains, dCas9 promoters, and guide RNA (gRNA) promoters. The CRISPRi tool was tested using nine gRNAs to target the promoters of MoATG3, MoATG7, and UvPal1. The results indicated that a single gRNA could direct the dCas9-fused transcriptional repression domain to efficiently silence the target gene in M. oryzae and U. virens. In both fungi, the target genes were repressed >100-fold, and desired phenotypes were observed in CRISPRi strains. Importantly, we showed that multiple genes could be easily silenced using polycistronic tRNA-gRNA in CRISPRi. Furthermore, gRNAs that bind different promoter regions displayed variable repression levels of target genes, highlighting the importance of gRNA design for CRISPRi efficiency. Together, this study provides an efficient and robust CRISPRi tool for targeted gene silencing in M. oryzae and U. virens. Owing to its simplicity and multiplexity, CRISPRi will be a useful tool for gene function discovery in fungal pathogens. IMPORTANCE Many devastating plant diseases are caused by fungal pathogens that evolve rapidly to adapt to host resistance and environmental changes. Therefore, genetic tools that enable fast gene function discovery are needed to study the pathogenicity and stress adaptation of fungal pathogens. In this study, we adopted the CRISPR/Cas9 system to silence genes in Magnaporthe oryzae and Ustilaginoidea virens, which are two dominant fungal pathogens that threaten rice production worldwide. We present a versatile and robust CRISPRi toolkit that represses target gene expression >100-fold using a single gRNA. We also demonstrated that CRISPRi could simultaneously silence multiple genes using the tRNA-gRNA strategy. The CRISPRi technologies described in this study would accelerate the functional genomics of fungal pathogens.

Project description:RationaleDSP (desmoplakin), the most abundant component of desmosomes, which maintain the mechanical integrity of epithelium, is a genome-wide association study-identified genetic risk locus in human idiopathic pulmonary fibrosis (IPF). Subjects with IPF express a significantly higher level of DSP than control subjects.ObjectivesDetermine potential mechanisms by which DSP is regulated in lung fibrosis.MethodsMatrigel-coated soft and stiff polyacrylamide gels were made to simulate the stiffness of normal and fibrotic lungs. Quantitative chromatin immunoprecipitation and electrophoretic mobility shift assay were used to evaluate transcription factor binding to the DSP promoter. Targeted DNA methylation was achieved by CRISPR (clustered regularly interspaced short palindromic repeats)/dCas9 (deactivated CRISPR-associated protein-9 nuclease)-mediated Dnmt3A (DNA methyltransferase 3A) expression under the guidance of sequence-specific single guide RNAs.Measurements and main resultsStiff matrix promotes DSP gene expression in both human and rodent lung epithelial cells as compared with soft matrix. A conserved region in the proximal DSP promoter is hypermethylated under soft matrix conditions and becomes hypomethylated/demethylated under stiff matrix conditions. Demethylation of this conserved DSP promoter region is associated with transactivation of transcription factor EGR1 (early growth response protein 1), resulting in EGR1-dependent DSP overexpression. Targeted DNA methylation by CRISPR/dCas9/Dnmt3A-mediated epigenome editing blocks EGR1 binding to the DSP promoter and inhibits stiff matrix-induced DSP overexpression.ConclusionsDSP is a matrix stiffness-regulated mechanosensitive gene. CRISPR/dCas9-Dnmt3A-mediated epigenome editing reverses DSP overexpression by reestablishment of the epigenetic control of DSP under the mechanically homeostatic environment. It provides a useful tool for investigations of the functional role of DSP in the pathogenesis of lung fibrosis.

Dataset Information

Effective Blocking of Microbial Transcriptional Initiation by dCas9-NG-Mediated CRISPR Interference.

Publications

Effective Blocking of Microbial Transcriptional Initiation by dCas9-NG-Mediated CRISPR Interference.

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