Optimized CRISPR-Cpf1 system for genome editing in zebrafish

Methods. 2018 Nov 1;150:11-18. doi: 10.1016/j.ymeth.2018.06.014. Epub 2018 Jun 28.

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.

Keywords: Cas12a; Cpf1; HDR; Temperature regulation; Zebrafish.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics*
  • Clostridiales / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • DNA End-Joining Repair / genetics
  • Endonucleases / genetics*
  • Gene Editing / methods*
  • Genome / genetics
  • RNA, Guide / genetics
  • Recombinational DNA Repair / genetics
  • Zebrafish / genetics*

Substances

  • Bacterial Proteins
  • RNA, Guide
  • Endonucleases