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Review
. 2020 Feb 21;16(8):1441-1449.
doi: 10.7150/ijbs.42299. eCollection 2020.

Engineering Cellular Signal Sensors Based on CRISPR-sgRNA Reconstruction Approaches

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Free PMC article
Review

Engineering Cellular Signal Sensors Based on CRISPR-sgRNA Reconstruction Approaches

Hengji Zhan et al. Int J Biol Sci. .
Free PMC article

Abstract

The discovery of the CRISPR systems has enriched the application of gene therapy and biotechnology. As a type of robust and simple toolbox, the CRISPR system has greatly promoted the development of cellular signal sensors at the genomic level. Although CRISPR systems have demonstrated that they can be used in eukaryotic and even mammalian cells after extraction from prokaryotic cells, controlling their gene-editing activity remains a challenge. Here we summarize the advantages and disadvantages of building a CRIRPR-based signal sensor through sgRNA reconstruction, as well as possible ways to reprogram the signal network of cells. We also propose how to further improve the design of the current signal sensors based on sgRNA-riboswitch. We believe that the development of these technologies and the construction of platforms can further promote the development of environment detection, disease diagnosis, and gene therapy by means of synthetic biology.

Keywords: CRISPR; riboswitch; sgRNA; signal sensor; synthetic biology.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Design of the CRISPR system for sensing light. The combination of CRISPR-dCas9 and optogenetics has led to the development of a gene transcriptional regulatory system based on CRISPR-dCas9 that regulates the expression of endogenous genes in response to light. An effective transcription complex can only be formed when exposed to blue light.
Figure 2
Figure 2
Design of the riboswitch for sensing molecules. The ribosome binding site (RBS) of the mRNA 5'UTR is blocked by its own antisense RNA. The RBS site can be exposed only when bound to a specific non-coding RNA(A) or a small molecule(B) , allowing the mRNA to be translated.
Figure 3
Figure 3
Previous types of CRISPR signal sensors for sensing specific proteins. (A) The P53-related signal sensor is unable to recognize mutated P53 proteins (MtP53) in cells, thus rendering the sensor silent in the P53-deficient cells. While the wild type P53 proteins (WtP53) specifically activate the P53-related signal sensor which would turn on the artificial genetic circuit to protect cells from damage. (B) The intracellular protein signal sensor mainly composed of two parts located in the cell membrane and inside the cell. One part is anchored to the membrane and fused at the C-terminus to the TEV cleavage site (TCS) which has already associated with a GAL4-VP16 transcriptional activator. The other part is fused to TEV protease (TEVp). Interactions between the two parts and the target proteins result in the TEVp-mediated release of membrane anchored GAL4-VP16 and output expression.
Figure 4
Figure 4
Previous types of CRISPR signal sensors based on sgRNA-riboswitch. (A) The additional corresponding sequence which contained aptamer-based riboswitch in the 3'-end of the sgRNA. (B) The additional corresponding sequence which contained aptamer-based ribozyme set in the 5'-end of the sgRNA.
Figure 5
Figure 5
Future designs of CRISPR signal sensors for detecting proteins of interest. (A) Original CRISPR-Cas9/Cpf1 versions work directly on DNA, but they cannot be regulated. (B) Ribozymes are added to additional corresponding sequences to participate in the aptamers-mediated regulation of sgRNAs. The interactions between Ligand and aptamer results in a structural change in the additional corresponding sequence, which activates the ribozymes to induce self-cleavage and thus the additional corresponding sequence was automatically released from the sgRNA.
Figure 6
Figure 6
Future designs of CRISPR signal sensors for detecting RNA molecules of interest. The detection of RNA molecules is accomplished by the specific trigger RNA, a corresponding antisense RNA complementary to the target RNA molecule. The trigger RNA can be inserted directly into the 3'-end of the sgRNA (A). Also, it can be effectively combined with ribozyme at the 3'-end of sgRNA (B).

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