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.

The selected primers will amplify a pegRNA that deletes Glicine in position 312 of GFP. This deletion can cause a non-fluorescent GFP protein. In lower case the sites generated for the gibson assembly of the pegRNA in the vector. :

• PEgRNA_GFP_312Gdel_F: gtcctaggtataatactagtCTATATCTTTCAAAGATGACgttttagagctagaaatagc
• PEgRNA_GFP_312Gdel_R:gggcccaagcttcaaaaaaaATCTTTCAAAGATGACGGAACTACAAgcaccgactcggtgccactt

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

The image taken from the class presentation illustrates a possible sequence design for an insertion in the GFP sequence. Keeping the same PAM,spacer and PBS sequences, just the sequence to be retro transcribed by the RTT need to be changed. I proppose to make a deletion that changes the open reading frame, resulting in no GFP activity. The resulting reverse primer is:

gggcccaagcttcaaaaaaaTCACTACTCTGACCTAGGGTGTTCAAgcaccgactcggtgccactt