Team:MIT/Optimizations

• ABOUT US
  Team Attributions
• BACKGROUND
  Alternative Splicing CRISPR RNA Binding Proteins REST
• PROJECT
  Guides and ASOs mKate-FF4 2 Exon mKate HBG 3 Exon mKate HBG 3 Exon Dual Fluorescence
• LAB WORK
  Parts/Improved Parts Protocols Notebook Future Work
• MODELLING
• HUMAN PRACTICES
  Integrated HP Public Engagement Collaborations InterLab
• AWARDS
  HP Silver HP Gold Education & Public Engagement Model Parts Collection

Optimization Experiments

mKate, Ms2, and ASO Optimizations

Before testing the tiling of our ASO guides, we needed to determine the ideal amount of mKate, Ms2, and ASO required to see the effects of our system.

mKate Titration

As discussed earlier, ASOs are unstable in the cell. Therefore, we needed to ensure the proper ASO to reporter ratio. We suspected that our system would be able to affect the splicing of mKate up to a threshold, after which the mKate would saturate the system. These expected results can be seen below. The different colored lines corrispond to different transfection bins

In our experiment, we varied the amounts of mKate-ff4 from 10 to 500 ng using ASO3 and ASO2+. The ASO2+ 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)

mKate Titration for ASO3

mKate-ff4 reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU) for ASO3. The color of the line indicate the transfection bins of each result.

For the mkate titration for ASO3, we observe a standard mkate curve for all reporter concentration above 100 ng. Below 100 ng there was a disturbance in the normal curve, indicating that our system might be acting on the system.

mKate Titration for ASO2+

mKate-ff4 reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU) for ASO2+. The color of the line indicate the transfection bins of each result.

For the mkate titration for ASO2+, we observe a disturbance at 100 ng of reporter. This indicates that our system is affecting the red output at this concentration. While expected to see a consistent output of red below our saturation threshold, the results still hold for the 100 ng case.

Based on these results, we decided to transfect with 100ng of mkate-ff4 moving forward.

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.

mKate, Guides, and Cas13a Optimizations

Before testing the tiling of our Cas13a guides, we needed to determine the ideal amount of mKate and guide required to see an effect on our sytem.

mKate Titration

As discussed earlier, guides are unstable in the cell. Therefore, we needed to ensure the proper ASO to reporter ratio. We suspected that our system would be able to affect the splicing of mKate up to a threshold, after which the mKate would saturate the system. These expected results can be seen below. The different colored lines corrispond to different transfection bins

In our experiment, we varied the amounts of mKate-ff4 from 10 to 500 ng using Guide2. The guide was 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)

mKate Titration for Guide2

mKate-ff4 reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU) for Guide2. The color of the line indicate the transfection bins of each result.

We observe a standard mkate curve for all reporter concentration above 100 ng. At 100 ng there was a disturbance in the normal curve, indicating that our system might be acting on the system.

Based on these results, we decided to transfect with 100ng of mkate-ff4 moving forward.

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