Three ways to design a tag insert

The pegRNA pipeline designs guides for adding a tag to the C-terminus of a protein by prime editing. Which strategy it uses depends on how big the insert is, and the person running it picks the mode.

• PE7, for inserts up to 40 bp: one pegRNA plus one nicking guide on the opposite strand.
• TJ-PE, for inserts up to about 500 bp: a tethered-junction design.
• Prime Assembly, for anything bigger: two pegRNAs whose flaps anneal to the ends of a linear donor.

Where it stands

PE7 and TJ-PE are the working modes. Prime Assembly is a prototype and is not offered for real designs yet, because it cannot pair real efficiency predictions as built, so large inserts go through TJ-PE in the meantime. Efficiency predictions come from PRIDICT 2.0, and the runs happen on Imperial's CX3 cluster.