Up-to-date CRISPR knock-out protocol in zebrafish?

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.

Sofie H. Seibel

Hi Monika,

I also found the supplements of this article quite useful:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248249/

Ranjai Kumar

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

Bhavnesh Kumar

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.

Miguel Angel Moreno-Mateos

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,

Yang Yu

Dear Esteemed Colleague,

Greetings. I trust this message finds you well and eagerly pursuing advancements in the realm of genetic engineering, specifically within the context of zebrafish research. Your interest in an up-to-date CRISPR-Cas9 knock-out protocol for zebrafish signifies a commitment to leveraging cutting-edge methodologies for elucidating genetic functions and mechanisms in this model organism. Below, I provide a concise yet comprehensive guide to the current best practices for achieving successful CRISPR-Cas9 mediated gene knock-out in zebrafish.

Overview

CRISPR-Cas9 technology has revolutionized genetic studies in zebrafish, enabling precise and efficient gene editing. This method involves designing guide RNAs (gRNAs) to target specific genomic sequences, alongside the Cas9 nuclease, to induce double-strand breaks (DSBs) at desired loci. The cell's repair mechanisms, predominantly non-homologous end joining (NHEJ), then introduce insertions or deletions (indels) that can result in gene knock-out.

Protocol Steps

Target Site Selection:Utilize bioinformatics tools to identify and design gRNAs targeting the gene of interest. Ensure specificity to minimize off-target effects. Tools like CRISPRScan or CHOPCHOP can be invaluable for this purpose.

gRNA Synthesis:Synthesize gRNAs using in vitro transcription kits following manufacturer instructions. Purify the transcribed gRNAs to remove template DNA and transcription reagents.

Cas9 Preparation:Obtain Cas9 protein from a reliable commercial source. Alternatively, Cas9 mRNA can be synthesized in vitro for co-injection with gRNAs.

Microinjection into Zebrafish Embryos:Prepare zebrafish embryos for microinjection by collecting them immediately post-fertilization. Dilute the gRNA and Cas9 mRNA/protein to appropriate concentrations in injection buffer. A common practice involves using 12.5-25 ng/μL of each gRNA with 300-500 ng/μL of Cas9 mRNA or 100-300 ng/μL of Cas9 protein. Inject the gRNA-Cas9 mixture into the cell of one- to two-cell stage embryos using a microinjection setup. Inject approximately 1-2 nL per embryo.

Embryo Rearing:Post-injection, incubate the embryos at 28.5°C in E3 embryo medium, monitoring for normal development.

Screening for Mutagenesis:At the appropriate developmental stage, typically 24-72 hours post-fertilization, genotype embryos to identify those with successful gene knock-out. Techniques such as PCR followed by Sanger sequencing, restriction fragment length polymorphism (RFLP) analysis, or T7 endonuclease I assay can be employed.

Off-Target Analysis:Assess potential off-target effects using computational predictions followed by targeted sequencing of identified sites.

Considerations

Ethical Approval: Ensure all experiments are performed in accordance with institutional and national ethical guidelines regarding the use of zebrafish in research.
Optimization: Optimize injection concentrations and volumes for your specific setup and gene target to maximize efficiency and minimize toxicity.
Mosaic Analysis: Be aware of mosaicism in F0 generation zebrafish. Raising and breeding F0s to obtain F1 progeny can help in identifying germline transmission of edits.

By following this protocol and adapting as necessary for your specific gene target and research goals, you will be well-equipped to conduct CRISPR-Cas9 mediated gene knock-outs in zebrafish. This powerful approach continues to unlock new possibilities in genetic research, providing insights into gene function, developmental processes, and disease models.

Should you require further clarification, wish to discuss specific aspects of your project, or explore additional resources, please do not hesitate to reach out. I am here to support your scientific journey and contribute to the advancement of zebrafish genetics research.

Warm regards.

This protocol list might provide further insights to address this issue.

Help needed: How to map AA, AG, and GG genotypes on to polymorphism information?

Can anyone explain why my starters are amplifying sequence from human genome instead of HPV16?

Mounted sample stained with organic dyes (eg Alexa647) for superresolution?

No title

Can I add or delete studies from systematic review during revision stage of paper publication?

What is the glucose content in Cellic CTec2?

Paper authorship for students?

What is in common household dust?

I could not find a publication that fits the idea of evaporation together with DPD. Any suggestions?

How can I find out the molecular weight of acid chloride?

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

Anyone know of a good progesterone antibody?

What's the concentration recommend for recombinant protein use in western blotting?

Which polarizers are usually used to measure the birefringence of zebrafish muscles?

In what ways do fellow reaerchers think that mRNA vaccination may be under CRISPR systems technology application ?

Will SARS-CoV-2 Spike be broken into S1 and S2 by denaturing conditions?

Is there any free software for behaviour analysis of zebrafish ?

Kinetics of Enzyme production in Recombinant E Coli?

How can I completely delete a protein on the genomic level using Crispr?

Is it possible to quantify recombinant protein in the supernatant using SDS PAGE ?