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 .
