Part:BBa_K2429128:Experience

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Applications of BBa_K2429128

In order to test the effectiveness of the ASOs and guides in the family, we had to follow a series of steps to optimize the ratio of ASO/Guide, reporter, and protein. This process was best demonstrated with mKate-FF4 guides and ASOs.

ASO Titration

Once we had determined a proper amount of mKate-ff4 plasmid, we wanted to check whether increasing the amount of ASO plasmid in the cell would knockdown our mKate levels further. We expected that as the amount of ASO increased in the system, more ASOs would be available to target the mKate-ff4. After a certain amount of ASO was added, the system would become saturated with ASO and the knockdown would level-off. The expected results can be seen below. The different colored lines corrispond to different transfection bins

In our experiment, we varied the amounts of ASO1 and ASO1+ from 20 to 200 ng. The ASO1+ was co-transfected with Ms2, so that the Ms2 could bind to the hairpin loop attached to the ASO+. (For a detailed explanation of how to plan a mammalian transfection click here)

ASO Titration for ASO1

ASO plasmid amounts (20ng-200ng) vs. the amount of red fluorescence (AU) for ASO1. The color of the line indicate the transfection bins of each result.

There is no substantial change across ASO concentrations.

mKate Titration for ASO1+

ASO plasmid amounts (20ng-200ng) vs. the amount of red fluorescence (AU) for ASO2+. The color of the line indicate the transfection bins of each result.

There is no substantial change across ASO concentrations.

In both results there was no decrease across the ASO levels. We concluded that 200 ng was not enough to have an effect on our system. Therefore, in further experiments we increased the levels of ASO to 300 ng and saw better results.

Based on these results, we decided to transfect with 300 ng of ASO moving forward.

ASO Tiling

Our ASOs are tiled from the 3' splice site to the polypyrimidine stretch. In order to determine which site is most effective to target, we transfected all our ASOs under the same conditions, and compared their output to a non-targeting ASO. We expect that the non-targeting ASO will have no effect on the red output. We compare how much mKate each ASO knocks down to this standard. We expect there to be a variation between the ASOs.These expected results are shown below.

In our experiment, we tested four plain ASOs, ASO0 through ASO3, and five ASOs with hairpin loops, ASO0+ through ASO4+. ASO+s were co-transfected with Ms2. (For a detailed explanation of how to plan a mammalian transfection click here)

Plain ASO Tiling

ASO Type (ASO0-3, Junk ASO) vs. the amount of red fluorescence (AU).<

ASO 0, 1, and 3 had a knockdown in red from the control, with ASO3 showing the greatest decrease at about a log. ASO 2 had similar levels of red as the control.

ASO+ Tiling

ASO Type (ASO0+-4+, Junk ASO) vs. the amount of red fluorescence (AU).

The plain ASOs behaved as expected. Unexpectedly, the ASO+s had an increase in red from the control. Our hypothesis was that too much mKate was being produced before the ASOs and Ms2 had enough time to be translated. We did further experiments to add a time delay to the production of mKate.

Tre:mKate DOX Induction

ASOs and Ms2 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 ASO-affected output with a normal Tre:mKate-ff4 DOX induction. We expect that the ASOs 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 ASO1 and ASO1+, 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)

ASO1 Tre:mKate-ff4 DOX Induction                                          Plain Tre:mKate-ff4 DOX Induction

DOX Concentrations (5-500uM) vs. the amount of red fluorescence (AU) for ASO1 (left) and plain mKate(right). The color of the line indicate the transfection bins of each result.

Unexpectedly, the ASO condition has a higher red output than the plain mKate condition.

ASO1+ Tre:mKate-ff4 DOX Induction                                          Plain Tre:mKate-ff4 DOX Induction

DOX Concentrations (5-500uM) vs. the amount of red fluorescence (AU) for ASO1 (left) and plain mKate(right). The color of the line indicate the transfection bins of each result.

Unexpectedly, the ASO condition has a higher red output than the plain mKate condition.

Unfortunately, there was no significant difference between the control titration and the ASO or ASO+ inducton. All the ASOs will have to be tested in this way before determining whether or not this system is effective.

Guide Titration

Once we had determined a proper amount of mKate-ff4 plasmid, we wanted to check what concentration of guide plasmid was ideal for the Cas13a. Originally, our intuition told us that we wanted to maximize the amount of guide in the system as it degrades quickly. However, after reading a paper by Samira Kiani in which she used U6:guide constructs at 10ng, we reconsidered. We now expected to see results from the guide-Cas13a in values as low as 10 ng. The expected results can be seen below. The different colored lines corrispond to different transfection bins

In our experiment, we varied the amounts of Guide2 from 10 to 500 ng. Guide2 was co-transfected with Cas13a, 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 Titration for Guide2

Guide plasmid amounts (10ng-500ng) vs. the amount of red fluorescence (AU) for Guide2. The color of the line indicate the transfection bins of each result.

There was a decrease in red output when 10ng and above of guide is added to the system. Moving forward, we transfected with 10 ng of guide.

Based on these results, we decided to transfect with 10ng of guide moving forward. Now that we had optimized the different amounts for mKate-ff4, and guides, we were ready to test the effectiveness of our tiled guides

Guide Tiling

Our guides are tiled from the 3' splice site to the polypyrimidine stretch. In order to determine which site is most effective to target, we transfected all our guides under the same conditions, and compared their output to a non-targeting guide. We expect that the non-targeting guide will have no effect on the red output. We compare how much mKate each guide knocks down to this standard. We expect there to be a variation between the guides.These expected results are shown below.

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.

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