High-throughput Targeted Mutagenesis using CRISPR/Cas9 in Zebrafish

Monday, September 22, 2014 — Poster Session II
4:00 p.m. – 6:00 p.m.	FAES Academic Center	NHGRI	GEN-15

* FARE Award Winner

Authors

• G. K Varshney
• W Pei
• J Ledin
• M LaFave
• V Gallardo
• J Idol
• L Xu
• M Jones
• U Harper
• B Carrington4
• K Bishop
• M Vemulapalli
• M Li
• W Chen
• J Mullikin
• R Sood
• S. M. Burgess

Abstract

The zebrafish genome is now complete and is only the third vertebrate to have a fully annotated reference genome, which facilitates systematic large-scale functional genomic studies. The development of targeted mutagenesis approaches such as TALENs and CRISPR-Cas9 have opened up new avenues to mutagenize genome in a systematic fashion. The bacterial derived RNA-guided Cas9 endonuclease has emerged as a very powerful genome-editing tool in a wide variety of cells and organisms. We developed an inexpensive high-throughput method of multi-allelic targeted mutagenesis using the CRISPR-cas9 system. As a proof-of-principle, we targeted over 400 genes that included all zebrafish known or candidate orthologs for deafness in humans, kinases, genes involved in lateral line migration, and many genes involved in proteoglycan synthesis. By designing two targets per gene and using a high-throughput fluorescent PCR approach, we easily identified mutations at both targets individually as well as deletions of the regions between targets. We also generated a highly fecund lab strain NHGRI-1 and mapped all the possible polymorphisms in this line by deep sequencing. By having all polymorphisms identified, it allows us to computationally design CRISPR targets without additional target sequence validation.

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