Generation of Transgenic Fish Harboring CRISPR/Cas9-Mediated Somatic Mutations Via a tRNA-Based Multiplex sgRNA Expression

The controlled expression of Cas9 and/or sgRNA in transgenic zebrafish made it possible to knock out a gene in a spatially and/or temporally controlled manner. This transgenic approach can be more useful if multiple sgRNAs are efficiently expressed since we can improve the biallelic frame-shift mutation rate and circumvent the functional redundancy of genes and genetic compensation. We developed the tRNA-based system to express multiple functional sgRNAs from a single transcript in zebrafish and found that it is applicable to the transgenic expression of multiple sgRNAs. In this chapter, we describe a procedure for the generation of plasmids containing multiple sgRNAs flanked by tRNAs and a method to induce multiple CRISPR/Cas9-mediated genome modifications in transgenic zebrafish.