Knock-out mutagenesis of zebrafish genes using a CRISPR/Cas9 approach

Determining effective methods of shutting down genes or inserting a specific gene into the genome can provide insight about gene functionality and mechanisms for disease. My project specifically investigates methods of CRISPR/Cas9-mediated gene knock-out. I targeted two zebrafish genes, tram1 and clta. For tram1, I used one CRISPR/Cas9 mutagenesis site to generate loss-of-function alleles. For clta, I targeted two genomic sites around 140-bp apart to excise a portion of the chromosome. After raising several generations of fish, successful mutagenesis was confirmed. Analysis of genomic DNA showed tram1 mutant alleles with various insertions and deletions. Analysis of clta fish showed insertions and deletions as well as an allele with a 136-bp deletion. Results showed successful mutagenesis using both one- and two-target site approaches. The one-site approach proved to be an effective way of generating random mutations. The two-site approach proved to be an effective method of excising a portion of the genome.