I'm currently attempting to create a null mutation in a gene using CRISPR.

Here's what I've done:

1) Designed forward oligos for my gene of interest (GOI) and ordered a positive control, SLC (which causes an obvious white phenotype). I used CHOP CHOP to pick optimal sites BEFORE the catalytic domain in my GOI. I have a universal reverse that overlaps with the forward. Performed PCR with these primers, which serve as both template and primer. Ran gel with aliquot before PCR and after PCR to confirm everything worked as expected.

2) PCR clean up with kit

3) IVT with Takara (T7 Kit) and clean up with Trizol - ran on gel and saw a clean band for both GOI and SLC

4) Diluted RNA to 50 ng/uL and 100 ng/uL and saved some stock in 1 uL aliquots at -80 for injections. Also stored some undiluted stock just in case.

5) Injected into zebrafish embryos as soon as they mated to capture the single cell stage

6) At the end of 2 days post fertilization, I selected 10 pools of 5 embryos from my SLC injected and GOI. Also selected 1 pool from uninjected as a negative control. I should note, that roughly 94% of my SLC showed some degree of the phenotype, which made me really confident since my positive control was working great!

7) did DNA extraction and T7e on my fish - all of my SLC had cuts at expected but NONE of my GOI did :(

I cannot see anything obviously wrong with my technique - I've injected more concentrated guides into the embryos and got more deaths, and some very white SLC injected fish but my GOI injected embryos still do not work! I've cloned the PCR product from step 1 into a vector and sanger sequenced it to discover that it was exactly what I expected from CHOP CHOP. Also, I should note that I don't see a difference in death between my GOI injected pools and SLC injected pools, which makes me think this is not due to cutting an essential gene.

Researchers, I've done this with 4 different guides for my GOI and not a single one has worked for me. I'm so desperate for any insight. If anyone can offer any advice, I would greatly appreciate it.