Optimized CRISPR-Cpf1 system for genome editing in zebrafish.

Citation data:

Methods (San Diego, Calif.), ISSN: 1095-9130

Publication Year:
2018
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PMID:
29964176
DOI:
10.1016/j.ymeth.2018.06.014
Author(s):
Fernandez, Juan P; Vejnar, Charles E; Giraldez, Antonio J; Rouet, Romain; Moreno-Mateos, Miguel A
Publisher(s):
Elsevier BV
Tags:
Biochemistry, Genetics and Molecular Biology
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article description
The CRISPR-Cas9 system biotechnological impact has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient CRISPR-based 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.