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Optimized CRISPR-Cpf1 system for genome editing in zebrafish.

Juan P Fernandez1, Charles E Vejnar1, Antonio J Giraldez2

  • 1Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA.

Methods (San Diego, Calif.)
|July 3, 2018
PubMed
Summary

This study optimizes the CRISPR-Cpf1 (Cas12a) system for zebrafish genome editing. The protocol details efficient mutagenesis and homology-directed repair, crucial for genetic research in ectothermic organisms.

Keywords:
Cas12aCpf1HDRTemperature regulationZebrafish

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas systems offer powerful genome editing tools.
  • CRISPR-Cpf1 (Cas12a) is an efficient RNA-guided endonuclease.
  • Optimization is needed for specific model organisms like zebrafish.

Purpose of the Study:

  • To provide a detailed protocol for CRISPR-Cpf1 (Cas12a) system optimization in zebrafish.
  • To enable efficient genome editing and homology-directed repair in zebrafish.
  • To facilitate genetic modification in ectothermic organisms.

Main Methods:

  • In vitro design and generation of crRNAs.
  • Purification of recombinant Cpf1 (Cas12a) proteins.
  • Assembly of ribonucleoprotein (RNP) complexes for zebrafish mutagenesis.
  • Induction of Cpf1-mediated homology-directed repair using ssDNA oligonucleotides.

Main Results:

  • CRISPR-Cpf1 RNP complexes efficiently mutagenize the zebrafish genome.
  • crRNAs are unstable and degraded in vivo without Cpf1 protein.
  • Temperature significantly affects Cpf1 activity, with reduced performance below 37°C.

Conclusions:

  • The described protocol enables efficient and constitutive genome modification in zebrafish using CRISPR-Cpf1 (Cas12a).
  • This method facilitates homology-directed repair in zebrafish.
  • The protocol is applicable to other ectothermic organisms, expanding genome editing capabilities.