Gaurav K Varshney, Wuhong Pei, Matthew C LaFave, Jennifer Idol, Lisha Xu, Viviana Gallardo, Blake Carrington, Kevin Bishop, MaryPat Jones, Mingyu Li, Ursula Harper, Sunny C Huang, Anupam Prakash, Wenbiao Chen, Raman Sood, Johan Ledin, Shawn M Burgess
The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing...
July 2015: Genome Research