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Objectives To develop an in vitro method for rapid evaluation of the capability of a designed single guide RNAs (sgRNAs) to guide Cas9 nucleases to cleave target loci in mammalian cells. Results We constructed a Cas9/sgRNA plasmid with two SP6 promoters to simultaneously express Cas9 nuclease and the sgRNA and a negative selection plasmid harbouring a target site of the sgRNA. After co-transforming...
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