Hi everyone,
Im trying targeted gene editing in citrus protoplast through PEG-mediated transformation. Im using sgRNA+Cas9 RNP complex for DNA free genome editing. But im not successful in getting targeted mutation in target genes. Im using sgRNA synthesized using in vitro transcription kit and commercial SpCas9 (with NLS) from NEB. Before going for protoplast transformation i checked sgRNA+SpCas9 activity by in vitro digestion assay. Im getting efficient cleavage of target gene in that. But the same complex is not working in vivo (in protoplast). Any experienced researchers kindly post your suggestions that may help me to find out a solution. Send me any detailed protocol on this experiment if possible. Many thanks.
Check this one out for RNPs in Penicillium:
Genome Editing in Penicillium chrysogenum Using Cas9 Ribonucleoprotein Particles
Pohl, C., Mózsik, L., Driessen, A. J. M., Bovenberg, R. A. L. & Nygård, Y. I., May-2018, Synthetic biology: Methods and Protocols. Braman, J. C. (ed.). New York: Humana Press, p. 213-232 20 p. (Methods in Molecular Biology; vol. 1772).
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