The primary focus of the lab is to develop tools for studying gene function on a genome wide scale. We use 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 650 genomic locations and identified the mutations transmitted through the germline. We will continue to develop high-throughput techniques to generate approximately 1,000 more targeted gene KO's in the next 2 years. These mutations will feed into other ongoing projects in the lab. In addition, we are now focused on improving CRISPR-based alterations in the genome that are not specifically gene-inactivating, including targeted transgene integration and SNP knock-ins.

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Project End
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Budget End
Support Year
19
Fiscal Year
2019
Total Cost
Indirect Cost
Name
National Human Genome Research Institute
Department
Type
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Giannelou, Angeliki; Wang, Hongying; Zhou, Qing et al. (2018) Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors. Ann Rheum Dis 77:612-619
Tseng, Wei-Chia; Loeb, Hannah E; Pei, Wuhong et al. (2018) Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds. Dis Model Mech 11:
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Chandler, Randy J; LaFave, Matthew C; Varshney, Gaurav K et al. (2016) Genotoxicity in Mice Following AAV Gene Delivery: A Safety Concern for Human Gene Therapy? Mol Ther 24:198-201
Varshney, Gaurav K; Burgess, Shawn M (2016) DNA-guided genome editing using structure-guided endonucleases. Genome Biol 17:187

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