Journal
METHODS
Volume 150, Issue -, Pages 11-18Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2018.06.014
Keywords
Cpf1; Cas12a; Zebrafish; HDR; Temperature regulation
Funding
- Programa de Movilidad en Areas de Investigation priorizadas por la Consejeria de Igualdad, Salud y Politicas Sociales de la Junta de Andalucia
- NIH [R21 HD073768, R01 HD074078, GM103789, GM102251, GM101108, GM081602]
- Swiss National Science Foundation [P2GEP3_148600]
- Australian National Health and Medical Research Council [APP1090875]
- HHMI Faculty Scholar program
- March of Dimes
- Yale Scholars Program
- Whitman fellowship
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM081602, R35GM122580] Funding Source: NIH RePORTER
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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 muta-genize 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.
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