Zebrafish targeted mutagenesis and functional genomics

National Human Genome Research Institute

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Abstract

The primary focus of the lab is to develop tools for studying gene function on a genome wide scale. We have recently shifted our mutagenesis efforts from random retroviral integration mutagenesis to using the bacterial CRISPR/Cas9 gene-targeting technique. This provides us with a cost effective way to efficiently target gene KO's in zebrafish. In the past year we have developed a streamlined pipeline for gene targeting using CRISPR/Cas9 where we can use fluorescent PCR and the ABI capillary sequencer to rapidly identify insertions or deletions caused by CRISPR/Cas9 induced double stranded breaks. Using this pipeline we have already targeted over 300 genomic locations and identified the mutations transmitted through the germline. We will continue to develop high-throughput techniques to generate approximately 2,000 targeted gene KO's in the next 3 years. These mutations will feed into other ongoing projects in the lab.