|ZFIN ID: ZDB-PUB-170706-6|
CRISPR/Cas9-Directed Gene Editing for the Generation of Loss-of-Function Mutants in High-Throughput Zebrafish F0 Screens.
Shankaran, S.S., Dahlem, T.J., Bisgrove, B.W., Yost, H.J., Tristani-Firouzi, M.
|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|
|PubMed:||28678442 Full text @ Curr Protoc Mol Biol|
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.
ABSTRACTThe 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.
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