Is anyone working with CRISPR/Cas9 in zebrafish and has a detailed up-to-date protocol with high efficiency? I would like to create a knock out of my protein of interest, but so far none of my techniques have worked.
Many thanks!
Hi Monika, thanks for your answer in my question. The CRISPR protocol we use is based on this publication http://www.pnas.org/content/110/34/13904.full.pdf.
mRNA synthesis:
We synthesize it from pCS2-nls-zCas9-nls (addgene). It is linearized with NotI and mRNA is synthesized with the SP6 mMessage mMachine kit (ambion). Polyadenilation is done with the Poly(A) tailing kit from ambion. We precipitate the mRNA either with LiCl or Ammonium acetate.
guide RNA synthesis:
We used pDR274 as the expression vector of the gRNA. We linearize pDR274 with BsaI and then cloned a pair of oligonucleotides (Corresponding to the region you are targeting) with overhangs matching the overhangs in the plasmid. The construct is then electroporated into E. coli, purified, linearized with HindIII and gRNA is synthesized with the T7 megashort script kit from ambion.
30pg of gRNA and 150pg of mRNA are injected in 1 cell embryos in the yolk.
I would recommed you to make gRNA from pT7tyrgRNA. It targets the tyr gene in zebrafish so your embryos would lack pigmentation in their eyes if indels are produced. We used it for troubleshooting so that we would know that the CAS9 was working and make sure that if something was not working it was related to the target sites or the gRNA.
Hi Monika, thanks for your answer in my question. The CRISPR protocol we use is based on this publication http://www.pnas.org/content/110/34/13904.full.pdf.
mRNA synthesis:
We synthesize it from pCS2-nls-zCas9-nls (addgene). It is linearized with NotI and mRNA is synthesized with the SP6 mMessage mMachine kit (ambion). Polyadenilation is done with the Poly(A) tailing kit from ambion. We precipitate the mRNA either with LiCl or Ammonium acetate.
guide RNA synthesis:
We used pDR274 as the expression vector of the gRNA. We linearize pDR274 with BsaI and then cloned a pair of oligonucleotides (Corresponding to the region you are targeting) with overhangs matching the overhangs in the plasmid. The construct is then electroporated into E. coli, purified, linearized with HindIII and gRNA is synthesized with the T7 megashort script kit from ambion.
30pg of gRNA and 150pg of mRNA are injected in 1 cell embryos in the yolk.
I would recommed you to make gRNA from pT7tyrgRNA. It targets the tyr gene in zebrafish so your embryos would lack pigmentation in their eyes if indels are produced. We used it for troubleshooting so that we would know that the CAS9 was working and make sure that if something was not working it was related to the target sites or the gRNA.
Hi Monika,
I also found the supplements of this article quite useful:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248249/
I am agree with Carlos Galicia. In addition to his protocol you can try some easy one too. you can design sgRNA oligo in agiland and purchase commercially like from IDT. then you can make mRNA directly. Also in knockin, you just sequence to be insert and add 1 kb homologous each side by PCR and Inject sgRNA, CAS9 AND INSERT IN egg
HI Monika,
Two most important things in gene editing experiments are the selection of good gRNAs and good delivery system. If anyone of these two are selected poorly then experiment will fail. I would suggest to redesign your gRNAs and check their efficiency before delivery into target cells by in-vitro methods and second most important part delivery system and for this first try to search for the commercially available vector for your model system (I believe many options are now available) which have been reported to be successfully used and if you do not find any suitable one then you need to re-modify the conventional construct for same purpose by simple including Cas9 gene and its promoter in the expression cassette.
Best wishes and Good Luck.
Hi,
this is a step by step protocol for CRISPR-Cas9 in zebrafish we wrote past year:
http://cshprotocols.cshlp.org/content/2016/10/pdb.prot086850.full?sid=c40f9603-ec8a-4224-8e7f-38cb21ae3e20
Any question let me know.
Best,
M.
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