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. We have developed a streamlined pipeline for gene targeting using CRISPR/Cas9 where we use fluorescent PCR and the ABI capillary sequencer to rapidly identify insertions or deletions caused by CRISPR/Cas9 induced double stranded breaks. We can verify mutations using MiSeq data and specialized software we wrote. Using this pipeline we have already targeted over 500 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.
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