Analysis of scn5Laa and scn5Lab Gene Function in Danio rerio (Zebrafish) Heart Development through TALENs/CRISPR-CAS9-mediated Gene Knockout
Research in our laboratory is focused on understanding the molecular developmental genetic effects of crude oil exposure on vertebrate embryogenesis. Our research has demonstrated that exposure of zebrafish embryos to crude oil results in characteristic developmental anomalies marked by pericardial edema, progressive deterioration of the developing heart and cardiac stasis coupled with larval death (Ghiassi, 2014). Chopra et al. observed very similar developmental defects after antisense oligonucleotide (MO)-mediated knockdown of the voltage-gated sodium ion channel genes scn5Laa and ab (Chopra et al. 2010). They further demonstrated that the developmental defects induced by the zebrafish scn5Laa and ab-directed MO's acted independently of their effects as ion channel disrupters, indicative that the scn5Laa and ab gene-encoded proteins have separate ion channel and developmental functions. Since knockdown of zebrafish scn5Laa and ab induce defects that effectively phenocopy the teratogenic effects of crude oil in the zebrafish model, we wanted to verify that the scn5Laa and ab genes function in normal zebrafish heart development. Since MO-mediated gene knockdown phenotypes are often associated with off-target MO activities, verification of the activity of these genes in development required gene knockouts. Therefore, the aim of this research was to create zebrafish scn5Laa and scn5Lab gene knockouts using both TALENs and CRISPR-CAS9 gene editing methods and to determine whether these knockout mutants mirror the phenotypes observed by Chopra et al. (Chopra et al. 2010). Assuming that the effects of MO's on translation of scn5Laa and scn5Lab gene-derived mRNA are specific and results in gene knockdowns that directly affect embryonic heart development, we hypothesized that the zebrafish scn5Laa and ab gene knockouts would generate developmental defects similar to those reported by Chopra et al. (Chopra et al. 2010). To test our hypothesis, we created homozygous zebrafish scn5Laa and ab frameshift mutants using both TALENs and CRISPR-CAS9 gene editing methods. Screening of independent homozygous scn5Laa and scn5Lab frameshift mutant alleles for developmental defects, failed to demonstrate an increase in embryos with defects above frequencies observed among appropriate wild-type zebrafish controls. Since Chopra et al. (2010) observed stereotypical development heart defects among zebrafish treated independently with MO's directed toward either scn5Laa or scn5Lab genes, we reject the hypothesis that independent scn5Laa or ab gene knockout can generate mutants with developmental heart defects that mirror those induced by MO's. The absence of developmental heart defects among zebrafish bearing frameshift mutations in either scn5Laa or ab suggest that these genes act independently are not responsible for specification of normal zebrafish heart development. It is, however, possible that the absence of a detectable heart defect among knockout mutants may reflect the fact that both paralogs are required for normal heart specification and that Chopra et al.'s (2010) MO treatment with individual MO's simultaneously affected the expression of both genes. If so, then double knockouts of scn5Laa and ab would be required to recapitulate the heart developmental defects reported by Chopra et al. (2010). It is also possible that other members of the Na+ ion channel family proteins are required for normal heart development and that Chopra et al.'s MO knocked down their activities sufficiently to induce developmental heart defects. If so, additional gene knockouts for additional members of the Na+ ion channel family genes would have to be constructed and tested separately or in combination to test this hypothesis. It is also possible that the Chopra et al. results were attributable to widely acknowledged MO off-target, non-specific effects. Future experiments including the screening of scn5Laa and ab double mutants, will provide information about whether both genes are required to specify normal heart development and whether the results reported by Chopra et al (2010) were related to simultaneous knockdown of scn5a paralogs by single MOs.
Chokshi, Payal. (January 2015). Analysis of scn5Laa and scn5Lab Gene Function in Danio rerio (Zebrafish) Heart Development through TALENs/CRISPR-CAS9-mediated Gene Knockout (Master's Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/4923.)
Chokshi, Payal. Analysis of scn5Laa and scn5Lab Gene Function in Danio rerio (Zebrafish) Heart Development through TALENs/CRISPR-CAS9-mediated Gene Knockout. Master's Thesis. East Carolina University, January 2015. The Scholarship. http://hdl.handle.net/10342/4923. February 23, 2019.
Chokshi, Payal, “Analysis of scn5Laa and scn5Lab Gene Function in Danio rerio (Zebrafish) Heart Development through TALENs/CRISPR-CAS9-mediated Gene Knockout” (Master's Thesis., East Carolina University, January 2015).
Chokshi, Payal. Analysis of scn5Laa and scn5Lab Gene Function in Danio rerio (Zebrafish) Heart Development through TALENs/CRISPR-CAS9-mediated Gene Knockout [Master's Thesis]. Greenville, NC: East Carolina University; January 2015.
East Carolina University