Methodology

Methodology

Our optimized protocol includes injection of a mixture of mRNA encoding the Streptococcus pyogenes Cas9 protein, guide RNA, and donor ssDNA (repair template) into zygotes. On average, from 50 zygotes injected, 10 pups were born, from which only 1 or 2 had the correct genome modification. For point mutations a 100 nucleotide long synthetic ssDNA is used as donor DNA. To integrate large constructs like the EGFP coding sequence we have developed an efficient procedure for generation of long ssDNA donors. A methodological publication describing our protocol is under preparation.             

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