Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish

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.

CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development

“…In contrast, recently, Ota et al (2016) have reported a similar strategy to knock-in eGFP at the same genomic locus for pax2a , but the knocked-in allele generated a pax2a mutant allele resembling the homozygous pax2a null allele, no isthmus ( noi ; Brand et al, 1996). Differences in bait construction might account for why our strategy did not result in null alleles, compared to Ota et al (2016).…”

Involvement of the centrosomal protein 55 (cep55) gene in zebrafish head formation

“…In cep55 uy25 mutant embryos (n = 7), the islet1-positive motor neurons in the spinal cord were detected to be normal, whereas islet1-positive cranial motor neurons in the brain were reduced and disorganized (Figure 6b,d). Next, we used the cep55 uy25 mutant possessing the transgene pax2-hs:EGFP, which widely visualizes some regions of the midbrain, cerebellar primordium and hindbrain (Ota et al, 2016). We found that the pax2-positive cells in the isthmocerebellar were reduced in the cep55 uy25 mutant embryos (n = 5) compared to wild-type embryos (n = 10; Figure S5).…”

“…The otx2 expression domain was narrow in the cep55 uy25 mutant embryo. Next, we used the cep55 uy25 mutant possessing the transgene pax2-hs:EGFP, which widely visualizes some regions of the midbrain, cerebellar primordium and hindbrain (Ota et al, 2016). In wild-type embryos (n = 8), GFP expression was detected in the motor neurons in the midbrain (nIII neurons and nIV neurons), in the branchiomotor neurons in the hindbrain (nV neurons, nVII neurons and nX neurons) and in the motor neurons of the spinal cord (Figure 6a,c).…”

Exogenous gene integration mediated by genome editing technologies in zebrafish

“…When the donor vector was co-injected with Cas9 mRNA and 2 sgRNAs (targeting for the pax2a locus and for the donor vector) into zebrafish embryos, the eGFP expression in some of the injected F0 embryos partially overlapped with the pax2a expression domains. 28 We established the transgenic line Tg[pax2a-hs:eGFP], showing that the eGFP expression in the heterozygous embryos completely overlaps the endogenous pax2a expression domains 28 (Fig. 4B (i)).…”

“…We have also reported that the NHEJ-mediated integration of the reporter gene into the start codon is useful to monitor the expression pattern of an uncharacterized gene (epdr1: ependymin related 1) in the heterozygous embryos and to analyze the loss-of-function phenotypes in the homozygous embryos. 28 Recently, Hoshijima et al have demonstrated reporter gene insertion just downstream of the start codon of the potassium voltage-gated channel subfamily H member 6a (kcnh6a) gene in zebrafish using TALEN system. 29 The kcnh6a gene is exclusively expressed in the heart, and its disruption causes a cardiac disorder.…”