CRISPR-based CopyCatcher Plasmid Assembly

One-step assembly with NEBuilder HiFi DAssembly Master Mix was used for cloning of CopyCatcher plasmids^{91 (link)}. As depicted in Fig. 1a, left and right homologous arms for each locus flanking gRNA cleavage site were amplified from the w¹¹⁸ wild-type fly genomic DNA. The SA-T2A sequence was amplified from pBS-KS-attB2-SA-T2A-3XGal80-Hsp70 series plasmids (Addgene 62951 and 62952)^{92 (link)}, gRNAs-expressing cassette targeting to the first intron of yellow, white, and ple were assembled following online published protocols (CRISPR fly Design-http://www.crisprflydesign.org/) and mCerulean selection marker was amplified from Addgene 27795 plasmid. DsRed was put at the downstream of T2A to indicate the copying events by fluorescence. Following assembly, CopyCatcher plasmids were transformed into NEB 5-alpha chemical competent E. coli cell (NEB C2987). Positive plasmids verified by sequencing were purified with Qiagen Plasmid Midi Kit (#12191), mixed with pBS-Hsp70-Cas9 (Addgene 46294) at 500 ng/μl and 250 ng/μl each, and sent to Rainbow Transgenic Flies, Inc. for injection into w¹¹⁸ wild-type (y^[CC] and ple^[CC]) or Oregon-R (w^[CC]) embryos. By screening the CFP fluorescent eye-marker phenotype, male transformants carrying the y^[CC], w^[CC] and ple^[CC] CopyCatcher elements were identified and followed by genomic insertion confirmation by flanking PCR^{19 (link)}.