Plasmid construction Primers used in this work were synthesis by Beijing Tsingke Biotech Co., Ltd (Tianjin, China), the gene fragments of EcCas6 and three toxins were synthesized by GenScript (Nanjing, China). The RfxCas13d plasmid and the shRNA vector were purchased from Miaoling Biotech Co., Ltd. EcCas6 and RfxCas13d fragments underwent PCR amplification using 2× Phanta UniFi Master Mix (Dye Plus) (P526, Vazyme Biotech Co., Ltd, Nanjing, China), then cloned into a pCMV vector via 2× MultiF Seamless Assembly Mix (RK21020, Abclonal Technology, Wuhan, China), and transformed into Trelief 5α chemically competent cell (DLC101, Beijing Tsingke Biotech Co., Ltd, Tianjin, China). To generate dEcCas6-toxin mutants, various toxin mutations were introduced via PCR and the amplification products were overlapped with the dEcCas6 fragment before being cloned into a pCMV vector.
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