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
