Abstract
The CRISPR/Cas9 complex, which is composed of a guide RNA (gRNA) and the Cas9 nuclease, is useful for carrying out genome modifications in various organisms. Recently, the CRISPR/Cas9-mediated locus-specific integration of a reporter, which contains the Mbait sequence targeted using Mbait-gRNA, the hsp70 promoter and the eGFP gene, has allowed the visualization of the target gene expression. However, it has not been ascertained whether the reporter integrations at both targeted alleles cause loss-of-function phenotypes in zebrafish. In this study, we have inserted the Mbait-hs-eGFP reporter into the pax2a gene because the disruption of pax2a causes the loss of the midbrain-hindbrain boundary (MHB) in zebrafish. In the heterozygous Tg[pax2a-hs:eGFP] embryos, MHB formed normally and the eGFP expression recapitulated the endogenous pax2a expression, including the MHB. We observed the loss of the MHB in homozygous Tg[pax2a-hs:eGFP] embryos. Furthermore, we succeeded in integrating the Mbait-hs-eGFP reporter into an uncharacterized gene epdr1. The eGFP expression in heterozygous Tg[epdr1-hs:eGFP] embryos overlapped the epdr1 expression, whereas the distribution of eGFP-positive cells was disorganized in the MHB of homozygous Tg[epdr1-hs:eGFP] embryos. We propose that the locus-specific integration of the Mbait-hs-eGFP reporter is a powerful method to investigate both gene expression profiles and loss-of-function phenotypes.
Highlights
Auer et al have reported the CRISPR/Cas9-mediated integration of reporter genes at targeted loci in zebrafish[11]
In the eGFP-positive F0 embryos (Fig. 1), we failed to detect the loss of the midbrain-hindbrain boundary (MHB) that occurs in the noi mutant, suggesting that the efficacy of the reporter knock-in was not sufficient to induce the noi phenotype
When pax2a-gRNA2 or pax2a-gRNA3 was co-injected with the Mbait-hs-eGFP reporter, Mbait-guide RNA (gRNA) and Cas[9] mRNA, the eGFP expression was detected in the MHB, the optic stalk and the hindbrain neurons
Summary
Auer et al have reported the CRISPR/Cas9-mediated integration of reporter genes at targeted loci in zebrafish[11]. The authors used the modified donor vector containing the Mbait sequence derived from the rat Mcr[4] gene, the hsp[70] promoter and the reporter/driver genes, and they demonstrated the CRISPR/Cas9-mediated locus-specific integration of the reporter, resulting in the continuous visualization of target gene expression. It remains uncertain whether the reporter gene integrations in both targeted alleles lead to loss-of-function phenotypes in zebrafish. We demonstrated that the locus-specific integration of the Mbait-hs-eGFP reporter by CRISPR/Cas[9] is a powerful genetic method for visualization of continuous target gene expression and for conducting a loss-of-function analysis of gene of interest in zebrafish
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