CRISPRi
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Recent papers in CRISPRi
CRISPR/Cas system, a microbial adaptive immune system, has rapidly transformed the ways researchers can interrogate the genome. CRISPR has many advantages over traditional methods such as Transcription activator-like effector nucleases... more
CRISPR/Cas system, a microbial adaptive immune system, has rapidly transformed the ways researchers can interrogate the genome. CRISPR has many advantages over traditional methods such as Transcription activator-like effector nucleases (TALEN) and Zinc-finger nucleases (ZFN). Since CRISPR discovery as an adaptive immune system used by bacterial against viruses, it has been repurposed to help in many different genome-related studies such as gene knocking in and out, gene expression upregulation and downregulation. Also CRISPR holds vast therapeutic potential for the management of genetic disorders by straight modifying disease-causing mutations. Although the Cas9 protein has been revealed to attach and cleave DNA at off-target sites, the field of Cas9 specificity is quickly progressing, with marked modifying in guide RNA choice, protein and guide engineering, innovative enzymes, and off-target recognition methods. In current review we mostly focus on CRISPR unique ability in gene activation/ upregulation, which has wide applications in different aspects such as gene studies, stem cell differentiation, and trans-differentiation. Compared to other gene activation methods such as viral gene overexpression, TALEN and ZFN, CRISPR offers many benefits such as easy designing and high precision.
Programmable control over an addressable global regulator would enable simultaneous repression of multiple genes and would have tremendous impact on the field of synthetic biology. It has recently been established that CRISPR/Cas systems... more
Programmable control over an addressable global regulator would enable simultaneous repression of multiple genes and would have tremendous impact on the field of synthetic biology. It has recently been established that CRISPR/Cas systems can be engineered to repress gene transcription at nearly any desired location in a sequence-specific manner, but there remain only a handful of applications described to date. In this work, we report development of a vector possessing a CRISPathBrick feature, enabling rapid modular assembly of natural type II-A CRISPR arrays capable of simultaneously repressing multiple target genes in E. coli. Iterative incorporation of spacers into this CRISPathBrick feature facilitates the combinatorial construction of arrays, from a small number of DNA parts, which can be utilized to generate a suite of complex phenotypes corresponding to an encoded genetic program. We show that CRISPathBrick can be used to tune expression of plasmid-based genes and repress chromosomal targets in probiotic, virulent, and commonly engineered E. coli strains. Furthermore, we describe development of pCRISPReporter, a fluorescent reporter plasmid utilized to quantify dCas9-mediated repression from endogenous promoters. Finally, we demonstrate that dCas9-mediated repression can be harnessed to assess the effect of down-regulating both novel and computationally-predicted metabolic engineering targets, improving the yield of a heterologous phytochemical through repression of endogenous genes. These tools provide a platform for rapid evaluation of multiplex metabolic engineering interventions.
- by Brady Cress and +3
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- Phytochemistry, Synthetic Biology, Probiotics, Systems Biology
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