The ASNA-1 (arsA arsenite transporter, ATP-binding, homolog 1) gene has been found to be involved in regulating insulin secretion in both C. elegans nematode and human pancreatic beta cells (Kao et al., 2007). Genetic knockout of ASNA-1 arrests growth in C. elegans and embryonic lethality in mice. Little is known about the ASNA-1 functions in vertebrate development. This project aims to study ASNA-1 using zebrafish embryos. We first tried to understand what cells or tissues express the ASNA-1 gene by identifying the zebrafish ASNA-1 cDNA sequence on ZFIN.org and designed primers to amplify ASNA-1 cDNA using PCR. We then purified the zebrafish ASNA-1 cDNA using gel extraction method followed by cloning cDNA into the pCRII-TOPO expression vector. Cloned cDNA was confirmed with restriction enzymes and later used to synthesize DIG-RNA probe for in situ hybridization. Our preliminary results suggest the ASNA-1 gene is expressed in the brain. In the meantime, we performed CRISPR to knockout the ASNA-1 zebrafish genes. We designed primers to target two exons of ASNA-1 gene that encode for the ATP binding site. We then used the primers to synthesize a DNA template and later guide RNA (gRNA). We have successfully synthesized three gRNAs targeting Exon 4 and 6. Currently, we are conducting micro-injection to deliver the gRNA and caspase 9 into embryos. After injection of the embryos, we will extract the genomic DNAs and examine if deletion has occurred by PCR. With the cloned ASNA-1, cDNA and gRNA, we will be able to understand the functions of ASNA-1 in the future.
