Part:BBa_K2429023:Experience

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Applications of BBa_K2429023

In our experiment, we tested five plain guides, Guide1 through Guide5.The guides were co-transfected with dCas13a, which uses the guide to locate the target intron and block the spliceosome from recognizing the splice site. (For a detailed explanation of how to plan a mammalian transfection click here)

Guide Tiling

Guide Type (Guide1-5, Junk ASO) vs. the amount of red fluorescence (AU).< In the presence of dCas13a, there was minimal knockdown. The greatest knockdown occurred in the presence of Guide2. Our hypothesis about the reason we were not seeing the knockdown we expected was that too much mKate was being produced before the guides and dCas13a had enough time to be translated. We did further experiments to add a time delay to the production of mKate.

Tre:mKate DOX Induction

Guides and Cas13a need to be produced by the cell before they are able to affect the splicing of mKate-ff4. In order to add a time delay between when they were being produced and when mKate was being produced, we put mKate downstream of a DOX inducible promoter. DOX was added to cells 24 hours after the initial transfection, allowing the cells to undergo a doubling before producing mKate. We want to compare the results of the Cas-affected output with a normal Tre:mKate-ff4 DOX induction. We expect that the Cas will cause a disruption in what would otherwise be a normal mKate induction curve. These expected results are compared side by side below. The different colored lines corrispond to different transfection bins

In our experiment, we used Guide3, and a well with just mKate for control. DOX was added to the system 24 hours after transfection. Each cell was transfected with optimized amounts of plasmid from previous experiments (For a detailed explanation of how to plan a mammalian transfection click here)

Guide 3 Tre:mKate-ff4 DOX Induction                                          Plain Tre:mKate-ff4 DOX Induction

DOX Concentrations (5-500uM) vs. the amount of red fluorescence (AU) for Guide3 (left) and plain mKate(right). The color of the line indicate the transfection bins of each result.

Unfortunately, there was no significant difference between the control titration and the Guide3 or Junk guide induction. However, even the control in this experiment was not showing clear results, so the experiment should be rerun before drawing conclusions about the system. All the guides will have to be tested in this way before determining whether or not this system is effective.

Lethbridge_HS iGEM 2019

This protein is an RNA cleaving enzyme called Cas13a that was isolated from Leptotrichia shahii (Lsh). It interacts with a direct repeat stem loop on a CRISPR RNA (crRNA) that contains a specific sequence for a target RNA transcript. This construct is designed to be able to be expressed under an inducible promoter (T7). The protein itself has several purification tags (His, maltose binding protein tags, and a TEV site for cleaving purification tags off). This construct was ordered from Addgene.

Both parts of our system rely on the protein Cas13a, so it was imperative that we purify the protein for future use in our Cas13a activity assay. Initially, our team tried to express Cas13a in E. coli BL21 DE3; however, we had issues with overexpression so we tried again using E. coli Rosetta DE3 cells with greater success (Figure 1).

Figure 1. 10% SDS PAGE of overexpressed Cas13a proteins. Left to right: lane 1: Lba before induction; lane 2: Lba after induction; lane 3: Lbu before induction; lane 4: Lbu after induction; lane 5: Lsh before induction; lane 6: Lsh after induction; lane 7: Lwa before induction; lane 8: Lwa after induction; Lane 9: 10-250 kDa Protein Ladder (BioBasic); Lane 10: empty.

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