Seamless editing of the C. elegans genome using CRISPR/Cas9
Design the sgRNAs and order the primers (see the guidelines).
Clone the sgRNAs in pDD162 using Q5 mutagenesis kit (NEB).
Mix together the following: Prepare a negative control mix without the Forward primer.
Digest away pDD162 for 5min at RT with: 1ul of Q5 PCR 5ul of KLD 2X buffer 1ul of KLD 10X enzyme 3ul of H2O.
Add 5ul of the digested reaction to 50ul of kit‐provided competent cells. 
Heat shock at 42°C for 30s. 
Add 950ul of SOC medium and shake for 1h at 37°C.
Plate 25ul on Carb plate.
Centrifuge the remaining 975ul for 3min at 5K and also plate the pellet.
One half (or more) of the colonies will have the correct insertion.
Pick 6 colonies to grow each in 2ml of bacterial culture.
Miniprep (Qiagen kit, include the PB wash, elute in 50ul of H2O) and send to sequencing using the forward sequencing primer in the guidelines.
Design and order the repair ssODNs as described in the guidelines.
Reconstitute oligo at 1ug/ul according to the amount provided by the manufacturer.
Amplify the GFP plasmid pCM1.53 (available at Addgene) with primers containing the desired flanking regions (~30‐60 bp), mutations in the sgRNA site(s) and GFP sequence.
PCRs are performed using Phusion taq 2X Master Mix (NEB), 45s elongation step, 30 cycles, 50ul reaction.
Annealing step is done using a gradient from 60°C to 72°C.
Run PCR reactions on agarose gel to confirm the amplification. 
Pool positive PCRs (typically three reactions) and purify using a minelute PCR purification kit (Qiagen, elution with 10ul of H2O).
Pool the reactions and purify them using one minelute PCR purification column and measure the concentration.
The DNA concentration should be >500ng/ul (at this concentration, the amount of PCR oligo remaining in the mixture will be low enough to avoid any toxicity).
We use pRF4 roller plasmid at 120ng/ul, but you can use any marker that you find convenient.
Miniprep from 3ml of bacterial culture, as for the Cas9/sgRNA plasmid.
Do not let cultures grow for more than 16 hours.
Add H2O to 15ul Centrifuge at 13K for 15min on tabletop centrifuge.
Load injection needles with the injection mix.
Bleach a large plate of worms 
WashA: wash with M9.
WashB: wash with M9.
Plate embryos (less than 2000) on NA22 large plate.
(NOT completely covered with NA22 bacteria) Incubate multiple plates at different temperatures to ensure to have at least one with young adults (few embryos /one embryo row) on the day of injection.
Pick hermaphrodites with a sharp pick from areas of the plate where there are no bacteria and place on injection pad.
Inject 30‐40 worms.
AddingM9 #1: add 5ul of M9 (Start adding M9 every 5-10 minutes, about 1h after the worms have been put in recovery buffer).
AddingM9 #2: add 5ul of M9.
AddingM9 #3: add 5ul of M9.
AddingM9 #4: add 10ul of M9. 
AddingM9 #5: add 10ul of M9.
AddingM9 #6: add 15ul of M9.
AddingM9 #7: add 15ul of M9.
AddingM9 #8: add 15ul of M9.
AddingM9 #9: add 20ul of M9.
AddingM9 #10: add 20ul of M9. 
Put a drop of 20ul of M9 on a new OP50 plate, outside the bacteria layer.
With a pick, transfer 5 to 10 injected worms from the recovery buffer to the M9 drop and push them away from the M9 drop towards the food.
Repeat until all the worms are transferred.
Even if the worms look inert at this or the next step, they are worth transferring as they may yield edited progeny.
Leave the injected worms on OP50 plates at room temperature for 5h and then transfer each worm (P0) to a new OP50 plate (1 P0 per plate).
Allow the P0s to lay eggs at 20°C for 1 or 2 days.
Transfer the P0s to fresh OP50 plates between the first and second day.
Let the F1s grow at 20°C.
When all the F1s have reached the young adult stage (4 days at 20°C), check for rollers.
GFP fusions: if you know what you are looking for, it is possible to screen directly for GFP expression in the F1 (or F2) animals.
PCR screening: Transfer the F1 rollers and their non‐roller F1 siblings to new plates (2 to 8 F1s per plate).
PCR Screening: Let the F1 lay eggs for 24h at 20°C.
Lyse the F1s for PCR: In each 10 uL tube of lysis buffer, put 2 to 8 F1s.
We recommend testing each gene‐specific PCR assay before starting the injections.
See the guidelines for PCR Screening details.
Clone the F2/3s from positive F1 plates.
It is useful to let the worms crawl on a no‐bacteria plate before picking to avoid accidental transfer of siblings.
If 2 F1s were pooled per plate, clone 16 F2s.
If 8 F1s were pooled per plate, chunk the starved plate if necessary and clone 24 to 32 F2/F3s.
Lyse and PCR F2/3s using the same methods as for the F1s.
EXCEPT: When looking for homozygous GFP worms, use primers that flank the GFP fusion.
Use the PCR product for sequencing: Clean 25ul of the PCR reaction using Qiagen Minelute kit, elute with 10ul of H2O.
Use 7ul for this elution as a template and use a primer inside the PCR product for sequencing.
Once a homozygous F2/3 plate is identified, it is recommended to clone 4 worms again to new plates and to verify their genotype to ensure that the line is truly homozygous.
Freeze the worms.
We recommend freezing at least two independent lines (derived from different P0s if possible or different F1s) for each type of edit.
