Generation of Sulf1{KO; mCherry} by homologous recombination using the CRISPR/Cas9 system

Sulf1{KO; mCherry} was generated by CRISPR/Cas9-mediated homology-directed repair. The synthesized sequences were annealed and ligated into the BbsI-digested pU6-BbsI-chiRNA plasmid (a gift from Melissa Harrison, Kate O'Connor-Giles and Jill Wildonger, University of Wisconsin–Madison, Madison, WI; Addgene #45946, Cambridge, MA) (Gratz et al., 2013). To generate the repair template, an mCherry-coding sequence and the 1.2-kb homologous sequences on either side of the predicted DSBs were cloned into the pHD-DsRed-attP backbone (a gift from Melissa Harrison, Kate O'Connor-Giles and Jill Wildonger; Addgene #51019) (Gratz et al., 2014) using Gibson Assembly Master Mix (E2611S, New England Biolabs, Ipswich, MA). The mCherry-coding sequence was amplified by PCR from pUAST-mCherry (a gift from Thomas Neufeld, University of Minnesota, MN) (Chang and Neufeld, 2009). The 1.2-kb Sulf1 homology arms were amplified from genomic DNA of the vasa-Cas9 strain. A mixture of pU6-sgRNAs and the repair template was injected into the vasa-Cas9 embryos by BestGene Inc. (Chino Hills, CA). The homologous recombinants were screened by PCR and verified by Sanger sequencing.