CRISPR/Cas9-mediated genome editing in sea urchins

Abstract The CRISPR(clustered regularly interspaced short palindromicrepeat)/ Cas9( CRISPR-associated nuclease 9) technology enables rapid, targeted, and efficient changes in the genomes of various model organisms. The short guide RNAs(gRNAs) of the CRISPR/ Cas9system can be designed to recognize target DNA within coding regions for functional gene knockouts. Several studies have demonstrated that the CRISPR/ Cas9system efficiently and specifically targets sea urchingenes and results in expected mutant phenotypes. In addition to disrupting gene functions, modifications and additions to the Cas9protein enable alternative activities targeted to specific sites within the genome. This includes a fusion of cytidine deaminaseto Cas9( Cas9-DA) for single nucleotide conversion in targeted sites. In this chapter, we describe detailed methods for the CRISPR/ Cas9application in sea urchinembryos, including gRNA design, in vitro synthesis of single guide RNA(sgRNA), and the usages of the CRISPR/ Cas9technology for gene knockoutand single nucleotide editing. Methods for genotyping the resultant embryos are also provided for assessing efficiencies of gene editing.