Team:UNESP Brazil/Parts



     For the 2021 competition, our team described the following part: BBa_K3156891. It was originally developed by Tingzhen Liu of the iGEM19_SHSBNU_China group (2019-10-21) and refers to the CDS of SpdCas9.

     The Cas9 protein of the CRISPR-Cas type II system harbors two nucleolytic domains and the introduction of point mutations in the D10A and H840A domains blocks the nucleolytic activity of Cas9, losing its ability to generate a double-stranded DNA break, being named dead Cas9 or dCas9. However, its gRNA-guided target binding capacity remains the same. Thus, once the enzyme can reach the target gene, it can carry out its transcriptional control. However, high concentrations of dCas9 can be toxic to the cell, which can be a problem in the regulation of genetic circuits, as the protein needs to be always present. To minimize the use of the dCas9 protein and optimize regulatory processes, CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems were created.

     Registry Link: BBa_K3156891


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     To increase gene expression, our team decided to improve the part BBa_K3156891 (SpdCas9), to create the CRISPRa system. For this, we fused the gene of the SpdCas9 protein with that of the AsiA protein (Audrey Stevens’ inhibitor A), a 90 amino acid anti-σ70 protein found in the T4 bacteriophage. When fused to dCas9, this protein can increase gene expression up to 135 times (1). In the image below, it can be observed the CRISPRa system with AsiA managed to increase the strength of the J23117 (weak) promoter by 135 times, while other promoters such as J23116 (medium strength) and J23110 (strong) had the strength increased by 16 and 5 times, respectively.

Image 1. Increased promoter strength, related to increased fluorescence, compared to the negative control, after activation by the CRISPRa system. Adapted from: Ho et. al, 2020.

     Thus, we constructed the dCas9-AsiA gene to activate the production of fluorescent RNAs with the CRISPRa system, as shown in Figure 1 (response module figure), so that the strength of the promoters has increased and the RNAs produced only in the presence of the specific gRNA to the activation sites.

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     (1) Ho, H. I., Fang, J. R., Cheung, J., & Wang, H. H. (2020). Programmable CRISPR‐Cas transcriptional activation in bacteria. Molecular systems biology, 16(7), e9427.

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