CRISPR/Cas9‐Directed Gene Editing for the Generation of Loss‐of‐Function Mutants in High‐Throughput Zebrafish F0Screens
| Autor: | Martin Tristani-Firouzi, Sunita S. Shankaran, Timothy J. Dahlem, Brent W. Bisgrove, H. Joseph Yost |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Genetics biology ved/biology Cas9 ved/biology.organism_classification_rank.species Mutant biology.organism_classification Reverse genetics 03 medical and health sciences 030104 developmental biology 0302 clinical medicine Genome editing CRISPR Model organism Molecular Biology Zebrafish 030217 neurology & neurosurgery Loss function |
| Zdroj: | Current Protocols in Molecular Biology |
| ISSN: | 1934-3647 1934-3639 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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