I am injecting zebrafish with gRNA targeted to my gene of interest, a plasmid with that codes for a fluorescent protein, and gRNA targeted to the plasmid so that the plasmid inserts into my gene of interest around 1/3 of the way into the gene. This both knocks out the gene and inserts a fluorescent reporter. My positive control is a tyrosinase knockout which works well, and I also have another control where I inject rescue RNA at the same time to make sure the gRNA doesn't have any off target effects.
I am still looking for a negative control for CRISPR in zebrafish. I don't want to use scrambled because of off target effects and the fact they are poor controls. Can I inject crRNA that is unfused to the trRNA as a negative control? Is there a specific gene I could target?
I can think of two:
1. use a gRNA that target a human gene which does not recognized your GOI or any other zebrafish gene.
2. use a gRNA that have several mismatch compared to the one targeting your GOI. You may need do some readings on how many mismatch(s) would make you gRNA non-functional, i.e. cannot recognized your GOI.
I came up with these 2 ideas based on how controls for morpholino were designed. The first option is like the standard control morpholino that targets human Hb gene. The second option is like the 5-bp mismatch morpholino. I personally tried the first option as control for CRISPR knockout and it worked fine. I never tried it on CRISPR knockin like yours.
Hope this will be helpful.
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