Stop! Go! CRISPR as the Cellular Traffic Light

CRISPR (Clustered regularly interspaced short palindromic repeats) is a genetic tool that scientists use to edit DNA. The system consists of Cas9 proteins that cut out specific segments of DNA as instructed by guide RNAs (gRNA). To learn more about CRISPR, read one of our previous blog posts here.

Cells control which genes are transcribed and translated into proteins through a process called gene expression. Both eukaryotic and prokaryotic cells regulate gene expression, although they do so using different mechanisms. For example, in comparison to prokaryotic cells, eukaryotic cells have a lot more regulatory proteins that function to repress or activate transcription. Previously, scientists used CRISPR/Cas9 in eukaryotic cells to regulate gene expression. In short, they mutated the Cas9 protein (now called dCas9) so that it retained its ability to bind DNA but lost its ability to cut DNA. They then fused dCas9 to transcriptional regulators (proteins that regulate the copying of DNA into RNA) that either activated (CRISPRa) or repressed (CRISPRi) expression. (Overview a). The next step of the puzzle was to get the system working in prokaryotic cells.

Overview a

Overview b

Figure 1: a) CRISPR activation in bacteria enables complex multi-gene expression programs. Combining CRISPRa with CRISPRi enables multi-gene expression programs for simultaneous activation and repression of different target genes. Scaffold RNAs (scRNAs) that recruit activators can target genes for activation, while gRNAs targeted within a gene result in CRISPRi-based repression. If the CRISPR-Cas system components are controlled by inducible promoters, the entire gene expression program can be dynamically regulated. b) To activate gene expression, they target a CRISPR-Cas complex upstream of a target gene. dCas9 binds a scaffold RNA (scRNA), which is a modified gRNA that encodes both the target sequence and an RNA hairpin to recruit effector proteins that interact with RNA polymerase. The schematic depicts a 1x MS2 scRNA containing an MS2 RNA hairpin, which binds the MS2 coat protein (MCP) that is fused to candidate activator proteins.

In this article, researchers adapted the CRISPRa/i tool for the bacterium, Escherichia coli (E. coli). Instead of fusing Cas9 to transcriptional activators, they screened for proteins that could effectively activate gene expression when recruited by the CRISPR/Cas9 system. Having identified a strong activator protein, they demonstrated that they could simultaneously activate one target gene while repressing a different target gene. This suggests that pre-programmed gene expression programs could be simply switched on by inducing the expression of CRISPRa/i in a cell.

Bacteria are cost-effective and environmentally-friendly cell factories already in widespread use to produce biomedicine, food, material and energy. In the future, we will greatly improve biosynthetic yields by controlling branch points in metabolic networks with simultaneous gene activation and repression. Combined with RNA or protein-based biosensors that can sense and respond to cellular metabolic states, the gene activation tools developed by this research can be used to design more complex gene expression programs, which will be extremely beneficial for bacterial engineering. In conclusion, the researchers successfully developed a robust CRISPRa tool to enhance gene expression in bacteria. How soon could the technique be applied to other bacteria? Chances are we may not need to wait until next summer for an answer.

Summary written by: iGEM NEU_China_A team 2018

To read the full article, please click the following link:

Synthetic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria

Chen Dong, Jason Fontana, Anika Patel, James M. Carothers, Jesse G. Zalatan

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