Generating Cas9-mediated fluorescent protein knock-ins with a self-excising selection cassette (SEC)
See the Guidelines for: 1.
Choosing the Cas9 targets site 2.
Adding the target sequence to the Cas9–sgRNA construct 3.
Designing primers to add homology arms to an FP–SEC vector Grow bacteria carrying the FP–SEC vector and miniprep the plasmid DNA.
Note that, prior to replacing the ccdB elements with homology arms, FP–SEC vectors must be grown in cells that are resistant to ccdB (ccdB Survival cells).
Digest an entire miniprep of FP–SEC vector overnight at 37°C (consult Table P1 for which construct and enzymes to use).
Purify the digested vector using a PCR cleanup spin column to remove the enzymes.
Process the entire digested vector as one sample; do not attempt to gel purify individual bands.
The digested, purified vector may be stored at 4°C for at least a few months and reused to construct multiple repair templates.
Generate two PCR products (the homology arms) using genomic DNA as the template and the primers you designed above.
Mix the two PCR products and purify them together on a single PCR cleanup spin column.
Mix 1 µL of vector, 4 µL of homology arms and 5 µL of isothemal assembly enzyme mix (we use NEBuilder HiFi DNA assembly mix from NEB).
Incubate 1h @ 50°C or as directed by the enzyme manufacturer.
Transform 2 µL of the reaction to suitable competent cells.
Isolate DNA from 3-6 clones and sequence with M13 Forward and Reverse primers to verify correct insertion of the homology arms.
Prepare an injection mix containing the following: 10 ng/µL homologous repair template, 50 ng/µL Cas9-sgRNA construct with your targeting sequence Fluorescent co-injection markers (to label extrachromosomal arrays),  10 ng/µL pGH8 (Prab-3::mCherry neuronal co-injection marker; Addgene #19359), 5 ng/µL pCFJ104 (Pmyo-3::mCherry body wall muscle co-injection marker; Addgene #19328), 2.5 ng/µL pCFJ90 (Pmyo-2::mCherry pharyngeal co-injection marker; Addgene #19327). 
Prepare plasmid DNA using Invitrogen’s PureLink mini-prep kit, which gives high injection efficiencies.
Inject the mixture into the gonads of 50-60 young adult worms of strain N2 (or substitute any strain you like).
Transfer the injected worms to new seeded plates (three animals per plate works well in our hands).
Use regular NGM plates (no drug) at this stage.
Also make a control plate with uninjected worms, so that when you do the drug selection it can serve as a negative control.
Put the plates at 25°C and let the worms lay eggs without selection for 2-3 days.
Prepare and filter sterilize a 5 mg/mL hygromycin solution in water.
For 6 cm plates poured with 10 mL agar plates, pipet 500 µL of drug onto the surface of each plate of worms, for a final concentration of ~250 µg/mL (if using different size plates, adjust the volume accordingly).
Swirl gently so that the solution covers the entire surface of the plate, then let it dry.
Put the worms back at 25ºC.
Examine the plates and identify those that contain Roller (Rol) animals that survived the hygromycin treatment.
Candidate knock-in animals are L4/adults that 1) survive hygromycin selection; 2) are Rol; and 3) lack the red fluorescent extrachromosomal array markers.
Single 5-10 candidate knock-in adults to new plates without hygromycin.
Look for plates where 100% of L4s and adults are Rollers.
(See the Guidelines for details.)
Pick 6-8 L1/L2 larvae to each of three new plates.
It is possible to perform marker excision using older animals, but using young larvae results in the highest efficiency because the germ cells have not yet begun to divide.
Heat shock the plates at 34°C for 4 hours (or at 32°C for 4-5 hours) in an air incubator to activate expression of hs::Cre.
Return the plates to 20°C or 25°C.
Pick wild-type worms to new plates.
