Optimized CRISPR-Cpf1 system for genome editing in zebrafish
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 degrees 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.
|ISBN||1095-9130 (Electronic) 1046-2023 (Linking)|
|Authors||Fernandez, J. P.; Vejnar, C. E.; Giraldez, A. J.; Rouet, R.; Moreno-Mateos, M. A.|
|Responsible Garvan Author|
|URL link to publisher's version||https://www.ncbi.nlm.nih.gov/pubmed/29964176|