PUBLICATION
Optimized CRISPR-Cpf1 system for genome editing in zebrafish
- Authors
- Fernandez, J.P., Vejnar, C.E., Giraldez, A.J., Rouet, R., Moreno-Mateos, M.A.
- ID
- ZDB-PUB-180703-8
- Date
- 2018
- Source
- Methods (San Diego, Calif.) 150: 11-18 (Review)
- Registered Authors
- Fernandez, Juan Pablo, Giraldez, Antonio, Vejnar, Charles
- Keywords
- Cas12a, Cpf1, HDR, Temperature regulation, Zebrafish
- 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, Kinetoplastida/genetics
- Recombinational DNA Repair/genetics
- Zebrafish/genetics*
- PubMed
- 29964176 Full text @ Methods
Citation
Fernandez, J.P., Vejnar, C.E., Giraldez, A.J., Rouet, R., Moreno-Mateos, M.A. (2018) Optimized CRISPR-Cpf1 system for genome editing in zebrafish. Methods (San Diego, Calif.). 150:11-18.
Abstract
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping