Quantitatively Relating Gene Expression to Light Intensity via the Serial Connection of Blue Light Sensor and CRISPRi

Abstract

The ability to regulate endogenous gene expression is critical in biological research. Existing technologies, such as RNA interference, zinc-finger regulators, transcription-activator-like effectors, and CRISPR-mediated regulation, though proved to be competent in significantly altering expression levels, do not provide a quantitative adjustment of regulation effect. As a solution to this problem, we place CRISPR-mediated interference under the control of blue light: while dCas9 protein is constitutively expressed, guide RNA transcription is regulated by YF1-FixJ-PFixK2, a blue light responding system. With a computer-controlled luminous device, the quantitative relationship between target gene expression and light intensity has been determined. As the light intensifies, the expression level of target gene gradually ascends. This remarkable property enables sensor-CRISPRi to accurately interrogate cellular activities.

CRISPR-Cas9 Based Engineering of Actinomycetal Genomes

“…Besides the gene silencing effects of CRISPRi, it can also be extended to other applications. For instance, combining it with a blue light sensor allows for quantitative interrogation of cellular activities, and together with the CRISPR activators (CRISPRa) system it can enable systematic investigation of the cellular consequences of repressing or inducing individual transcripts, which can provide rich and complementary information for mapping complex pathways …”

“…Besides the gene silencing effects of CRISPRi, it can also be extended to other applications. For instance, combining it with a blue light sensor allows for quantitative interrogation of cellular activities, 50 and together with the CRISPR activators (CRISP-Ra) system it can enable systematic investigation of the cellular consequences of repressing or inducing individual transcripts, which can provide rich and complementary information for mapping complex pathways. 51 It is expected that the CRISPR-Cas9 gene deletion system and the CRISPRi based gene expression control system developed in this study will accelerate the metabolic engineering of actinomycetes, mutational/expression analyses of specific biosynthetic pathways, the in vivo generation of secondary metabolite derivatives, and the creation of genome minimized platform strains for heterologous expression of biosynthetic gene clusters (Figure S2).…”

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives

“…In the so-called CRISPR interference (CRISPRi) strategy (Gilbert et al, 2013), the cleavage-deficient dCas9 serves as a programmable repressor that can be adapted to near-arbitrary DNA targets via single-guide RNAs (gRNA) of matching sequence. This key property was exploited in several studies that regulate the expression of dCas9 in light-dependent manner, rather than its activity (Wu et al, 2014;Wu et al, 2021;Zhang and Poh, 2018). Although several directly light-regulated (d)Cas9 variants were developed for mammalian use, they often achieve optogenetic regulation via light-dependent recruitment of transcriptional effector modules but leave sequence-specific DNA binding, central to CRISPRi, unaffected by light.…”

Promoting microbiology education through the iGEM synthetic biology competition

“…Indeed, there was a noticeable boost in iGEM team publications in when the journal published several papers as part of an iGEM special issue (http://pubs.acs.org/toc/asbcd6/3/12). Several of these publications were particularly relevant to research involving bacteria (Atanaskovic, Bencherif, Deyell et al 2014;Buren, Karrenbelt, Lingemann et al 2014;Daszczuk, Dessalegne, Drenth et al 2014;Hendrix, Read, Lalonde et al 2014;Libis, Bernheim, Basier et al 2014;Nielsen, Madsen, Seppala et al 2014;Wang, Ding, Chen et al 2014;Wu, Wang, Cao et al 2014).…”

“…At the end of each competition each new BioBrick must be submitted to the iGEM Registry of Standard Biological Parts, from which it is made To assist exchange of BioBricks between teams and ensure there is transparency within the competition rules, each team must follow specific regulations that require cloning into specific backbones(Shetty, Endy and Knight 2008;Muller and Arndt 2012). However, even in iGEM projects complex gene assembly and editing approaches have been introduced, for example using Golden Gate cloning(Patron, Orzaez, Marillonnet et al 2015) and the CRISPR-Cas system(Wu, Wang, Cao et al 2014). There is a close relationship between iGEM and SynBio research, but the two are not synonymous.…”