Optimized CRISPR-Cpf1 system for genome editing in zebrafish.
Methods (San Diego, Calif.), Nov 2018PubMed: 29964176DOI: 10.1016/j.ymeth.2018.06.014

Optimized CRISPR-Cpf1 system for genome editing in zebrafish.

Fernandez JP,
Vejnar CE,
Giraldez AJ,
Rouet R,
Moreno-Mateos MA
  • Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA.
  • Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA.
  • Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT 06510, USA; Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06510, USA. Electronic address: antonio.giraldez@yale.edu.
  • Garvan Institute of Medical Research, Sydney, NSW, Australia. Electronic address: r.rouet@garvan.org.au.
  • Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA. Electronic address: moreno.mateos.ma@gmail.com.

Abstract

The impact of the CRISPR-Cas biotechnological systems has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient RNA-guided endonucleases. We have recently optimized CRISPR-Cpf1 (renamed Cas12a) system in zebrafish. We showed that (i) in the absence of Cpf1 protein, crRNAs are unstable and degraded in vivo, and CRISPR-Cpf1 RNP complexes efficiently mutagenize the zebrafish genome; and (ii) temperature modulates Cpf1 activity especially affecting AsCpf1, which experiences a reduced performance below 37 °C. Here, we describe a step-by-step protocol on how to easily design and generate crRNAs in vitro, purify recombinant Cpf1 proteins, and assemble ribonucleoprotein complexes to carry out efficient mutagenesis in zebrafish in a constitutive and temperature-controlled manner. Finally, we explain how to induce Cpf1-mediated homology-directed repair using single-stranded DNA oligonucleotides. In summary, this protocol includes the steps to efficiently modify the zebrafish genome and other ectothermic organisms using the CRISPR-Cpf1 system.
Product Used
Application
Bioengineering
Basic Research
Research Area
CRISPR Methods
Editing Method
Knock-in
Model Organism
Whole Animal
Paper Type
Peer-reviewed
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