Drosophila balancer reengineering using polycistronic gRNA for CRISPR/Cas9 gene editing
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| Gene Synthesis |
U6:3-tRNAHis -gRNAy,cv,v,f,car was synthesized by GenScript (Piscataway, NJ) and subcloned into pUASt-attB (Bischof et al. 2007) generating pU6:3-tRNAHis -gRNAy,cv,v,f,car . pU6:3-tRNAHis -gRNAy,cv,v,f,car was integrated into the P{CaryP}attP40 landing site (Markstein et al. 2008) through phi-C31 mediated integration (Bischof et al. 2007). Embryo injections for transgenesis and transformant recovery was completed by BestGene (Chino Hills, CA). Sanger sequencing was completed by Genewiz (Plainfield, NJ). Flies were raised on ‘Fly Food B’ (LabExpress, Ann Arbor, MI) at 25°C. A y – phenotype was scored by a reduction in pigmentation of the bristles for y 31d on FM7c. tRNAHis corresponds to tRNA:His-GTG-1-4 (FBgn0011878) and contains an additional six upstream nucleotides (GATGTA) from the annotated transcript. All sequences are available upon request. |
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