Seydoux lab Cas9 preparation test : Tag gtbp‐1 with eGFP or mCherry using dpy‐10 co‐CRISPR
Prepare the repair PCR template as indicated in Reagents for gtbp‐1 eGFP and mCherry tagging ( see guidelines ) and Repair template synthesis .
Make the injection mix as indicated in injection mixes , part I .
Use the crRNA gtbp‐1 Ct ( # 728 in Table S3 ) .
Inject 15‐20 young adult N2 worms and recover as described in Worm recovery and handling .
Pool the recovered worms ( P0s ) on one plate and incubate for 22‐23h at 20 °C ( day 1 ) .
Clone the P0s to individual OP50 plates and incubate at 20 °C for 4‐5 days ( day 2 ) .
When the F1s reach the adult stage , check for Rollers .
From those “jackpot broods” , screen the Rollers for fluorescent protein expression as described in Screening for edit of interest .
