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 .
