PgmNr Z6090: CRISPR-Cas9 based knock-in in zebrafish to facilitate streamlined visual genotyping.

Authors:
Roland S. Wu 1 ; Ian I. Lam 1 ; Thomas O. Auer 2 ; Filippo Del Bene 2 ; Shaun R. Coughlin 1


Institutes
1) Cardiovascular Research Institute and Department of Medicine, University of California San Francisco, San Francisco, CA; 2) Institut Curie, Centre de Recherche, Paris, France.


Abstract:

CRISPR/Cas9-based genome editing has greatly facilitated the ease of generating zebrafish genetic deletions. However, genotyping often requires significant investments in resources and remains a significant bottleneck in maintaining mutant zebrafish lines.  Furthermore, there are currently no broadly applicable methods to identify the genotype of a living, unperturbed zebrafish embryo. Here we describe a cloning-free genotyping system to facilitate convenient, high-throughput genotyping of living zebrafish mutant lines. We utilize a knock-in strategy using homology-independent DNA repair to insert a Gal4 reporter into an endogenous locus, allowing live interrogation of gene expression.  This knock-in harbors a cassette conferring lens fluorescence, and generation of mutant lines with lens fluorescence in different channels allows unambiguous genotyping of compound heterozygotes by visual examination of living embryos.  We use this system to disrupt the s1pr1 gene locus. Sphingosine-1-phosphate (S1P) has been found to play an important role in vertebrate development and effects of S1P are mediated by a family of five G protein-coupled receptors, sphingosine-1-phosphate receptors 1-5 (S1PR1-5), which confer the diversity of responses to S1P.  We have found that disruption of the s1pr1 gene leads to partial lethality in zebrafish larvae. Analysis of s1pr1 knock-in embryos reveals s1pr1 expression in the developing nervous system, caudal hematopoietic tissue, and vasculature.  We exploited live embryo genotyping to interrogate the effects of s1pr1 gene disruption in these tissues.  This strategy of gene disruption requires generating two new alleles instead of one per knockout and hence more initial investment than standard CRISPR/Cas9-based gene deletion. However, these knock-in lines allow visual genotyping of live embryos, greatly increasing the efficiency of maintenance and phenotypic evaluation, including identification of null embryos before the onset of phenotype.



ZFIN Genetics Index
1. s1pr1