PUBLICATION
CRISPR-Cas9 F0 knockout approach using predesigned in vitro transcribed guide RNAs partially recapitulates Rx3 function in eye morphogenesis
- Authors
- Wysocka, E., Gonicka, A., Anbalagan, S.
- ID
- ZDB-PUB-230202-15
- Date
- 2023
- Source
- Journal of genetics 102: (Journal)
- Registered Authors
- Anbalagan, Savani, Gonicka, Agata, Wysocka, Emilia
- Keywords
- none
- MeSH Terms
-
- Animals
- CRISPR-Cas Systems*
- Homozygote
- Larva
- Phenotype
- Zebrafish*/genetics
- PubMed
- 36722224 Full text @ J. Genet.
Citation
Wysocka, E., Gonicka, A., Anbalagan, S. (2023) CRISPR-Cas9 F0 knockout approach using predesigned in vitro transcribed guide RNAs partially recapitulates Rx3 function in eye morphogenesis. Journal of genetics. 102:.
Abstract
CRISPR-Cas9-based F0 knockout (KO) approach permits relatively simple and rapid generation of homozygous KOs and allows quick investigation of gene functions in zebrafish. However, F0 KO studies are largely performed using commercial synthetic guide RNAs (gRNAs) which are unaffordable by majority of the researchers. We tested (i) how effective is the CRISPR-Cas9-based F0 KO approach using in vitro transcribed gRNAs; (ii) how penetrant are the resulting phenotype at the later developmental stages and (iii) whether Coughlin's group pre-designed gRNAs are functional even without validating the gRNAs or testing for lack of SNP's in target loci. We targeted the rx3 gene that is required for the formation of the eye, a structure that exhibits robustness and can quickly recover from early phenotypes. Our results indicate that, in the majority of the samples, injection of Cas9 protein complex with four different in vitro transcribed gRNAs; targeting rx3 results in lack of eyes or disrupted eye development. Thus, the CRISPR-Cas9-based F0 KO approach using pre-designed, quadruple in vitro transcribed gRNAs can recapitulate the function of a gene at least until 5-dpf stage of larval zebrafish.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping