Direct delivery CRISPR - HDR editing protocol for C . elegans
Cas9 : : NLS can be purified from E . coli ( see our protocol ) or purchased from commercial sources .
Order tracrRNA and upon receipt , briefly spin the tubes .
Reconstitute tracrRNA at 4μg / μl ( 0.17nmol / μl ) : add 29.8μl of Tris pH 7.5 to the 5nmol provided ( U‐002000‐05 ) .
Order your gene specific crRNA ( see the guidelines for crRNA notes ) .
Upon receipt , briefly spin the tubes .
Reconstitute crRNA at 8μg / μl ( 0.6nmol / μl ) : add 33.8μl of Tris pH 7.5 to the 20nmol provided .
Use single‐stranded oligonucleotides ( ssODNs , 200nt maximum size , 4nM ultramer , salt free ) ordered from IDT .
( this is for small edits < 100nt ; see the guidelines for large edits 100bp - 2kb ) Reconstitute ssODN at 1μg / μl according to the amount provided by the manufacturer .
( this is for small edits < 100nt ; see the guidelines for large edits 100bp - 2kb ) Add each component of the injection mix in a 0.5ml tube ( add Cas9 last ) .
[ this if for " one locus editing " ] Place the 0.5ml tube in a 1.5ml eppendorf tube and spin for 2min at 13000rpm .
Incubate at 37°C for 10‐15min Immediately load the injection needles and process to injection .
Inject both arms of young adult hermaphrodites ( with a few embryos ) .
Recovery A .
Add 1X M9 .
5μl ( 30min to 1h after injection , recover the injected hermaphrodites ( P0s ) as follows : Every 5‐10min , add : 5μl / 5μl / 10μl / 10μl / 20μl / 20μl / 40μl of 1X M9 )
Recovery B : Add 1X M9 5μl .
Recovery C : Add 1X M9 : 10μl .
Recovery D : Add 1X M9 : 10μl .
Recovery E : Add 1X M9 : 20μl .
Recovery F : Add 1X M9 : 20μl .
Recovery G : Add 1X M9 : 40μl .
Clone out the P0s onto NNGM plates ( 1 P0 per plate , at 20°C ) .
Use fresh NNGM plates with a thin layer of OP50 bacteria at the center to facilitate screening for Rollers .
It is important to avoid that the P0s touch the mineral oil on the injection pad because the oil will kill them .
Transfer the P0s to second plate ( again 1 P0 per plate ) .
Examine the F1s for Rollers 4‐5 days after cloning the P0s .
Determine the number of Roller F1s per P0 and select the 3 P0s giving the most Rollers .
These are your “jackpot broods” .
Clone all the Roller F1s ( from the jackpot broods or from all broods if you do not have that many ) .
See the guidelines for : - Screening - Strain establishment - Special applications of this protocol
