PUBLICATION
CRISPR/Cas9-Directed Gene Editing for the Generation of Loss-of-Function Mutants in High-Throughput Zebrafish F0 Screens.
- Authors
- Shankaran, S.S., Dahlem, T.J., Bisgrove, B.W., Yost, H.J., Tristani-Firouzi, M.
- ID
- ZDB-PUB-170706-6
- Date
- 2017
- Source
- Current protocols in molecular biology 119: 31.9.1-31.9.22 (Chapter)
- Registered Authors
- Bisgrove, Brent, Shankaran, Sunita Sathy, Yost, H. Joseph
- Keywords
- CRISPR/Cas9, FO screen, gene editing, loss-of-function, recessive mutant, zebrafish
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems*
- Gene Editing/methods*
- Gene Knockout Techniques/methods*
- Genetic Testing/methods*
- Zebrafish/genetics*
- PubMed
- 28678442 Full text @ Curr Protoc Mol Biol
Citation
Shankaran, S.S., Dahlem, T.J., Bisgrove, B.W., Yost, H.J., Tristani-Firouzi, M. (2017) CRISPR/Cas9-Directed Gene Editing for the Generation of Loss-of-Function Mutants in High-Throughput Zebrafish F0 Screens.. Current protocols in molecular biology. 119:31.9.1-31.9.22.
Abstract
The ability to perform reverse genetics in the zebrafish model organism has been greatly advanced with the advent of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated) system. The high level of efficiency in generating mutations when using the CRISPR/Cas9 system combined with the rapid generation time of the zebrafish model organism has made the possibility of performing F0 screens in this organism a reality. This unit describes a detailed protocol for performing an F0 screen using the CRISPR/Cas9 system in zebrafish starting with the design and production of custom CRISPR/Cas9 reagents for injection. Next, two approaches for determining the efficiency of mutation induction by the custom CRISPR/Cas9 reagents that are easily performed using standard molecular biology protocols are detailed. Finally, screening for F0 induced phenotypes using the zebrafish flh gene as an example is discussed. © 2017 by John Wiley & Sons, Inc.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping