I genuinely could not follow what was being asked of these questions.

Genome Write & Edit Assignment

Design Assignment:

  1. Choose a pair of PEgRNA_GFP_F (forward) and PEgRNA_GFP_R (reverse) from the paper’s supplementary table 2. Explain what will be the edit and why you chose it.

  2. Design your own edit (RTT) using the same 20bp spacer and PBS.

  3. Bonus: Design your own set of forward and reference primers to introduce a different edit to the GFP domain. Use a different spacer and PBS.

<aside> ⚠️ Added Friday, April 19. Optional Bonus questions.

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Design a different pegRNA construct to introduce a different insertion, deletion, or substitution into the GFP gene. Some examples include:

Insertion

  1. Design a pegRNA to insert a small sequence (up to 33bp) in the GFP coding sequence that may affect the protein’s properties such as stability or fluorescence intensity.

Deletion

  1. Design a pegRNA to remove a portion of the GFP coding sequence (up to 97 bp) that may disrupt the proper folding or function of the protein, leading to a loss of fluorescence.

Substitution

  1. Design a pegRNA to introduce point mutations to increase brightness such as mutations in the superfolder GFP variants.
  2. Design a pegRNA to change chromophore-forming amino acids (chromophores are a part of the molecule responsible for its color) to potentially alter the fluorescence color.

PEgRNA → Prime Editing Guide RNA