Construction of a zebra fish knock-out mutant lacking a conserved bacterial homolog, yfiH

Abstract

YfiH gene codes an uncharacterized conserved protein widely distributed in 37 species from bacteria to human. The function of yfiH gene is still unknown, but preliminary data from Brevibacterium lactofermentum shows that the gene is not essential for the growth and viability. In vertebrates, the yfiH homolog is termed as LACC1. LACC1 encodes the enzyme laccase (multicopper oxidoreductase) domain; no precise information is there about this gene except that it has some implications in some autoimmune and autoinflammatory conditions in human like Rheumatoid arthritis, Juvenile idiopathic arthritis, Crohn’s disease and also leprosy.To know functional aspect of this gene it was knockout from zebrafish using CRISPR/Cas technique. CRISP Repeats are components of an immune system which protects many bacteria and archaea against foreign genetic elements. They function by targeting these elements in a sequence specific fashion, guiding the nuclease Cas9 to degrade them. The CRISPR-Cas system from S. pyogenes has been adapted and is used as an in vivo genome editing tool in a variety of organisms like zebrafish, mouse, and monkey and so on. In this project, we used it to knock out (KO) our gene of interest (LACC1) in zebrafish. This was achieved by co-injecting a genespecific single guide RNA (sgRNA) and Cas9 nuclease mRNA in early zebrafish embryos.Cas9 is RNA guided nuclease and creates double stranded break at the site where the single guide RNA bind. The cleaved DNA is repaired by Non Homologous End Joining (NHEJ) repair mechanism, generating unpredictable indel mutations. The induced mutations were detected by performing T7 endonuclease assay and were further confirmed by sequencing the target exons of the LACC1 gene. The exon 1 of LACC1 gene was successfully mutated using CRISPR-Cas9 technique. The guide RNA and Cas9 injected fishes were with deformed body with elongated body with curved tail and deformed head, showing the rate of deformity 6 in per 10 injected larvae. The phenotypes of the LACC1-deficient zebra fish mutants have to be further confirmed. Keywords: CRISPR Cas9, sgRNA, knockout, yfiH, LACC1.