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What is the benefit to using Cas9 protein or Cas9 mRNA instead of Cas9 plasmid or a Cas9-integrated cell line for HDR experiments?

Specifically, for HDR-mediated knockin experiments, Cas9 protein and Cas9 mRNA are preferred as they typically give higher gene editing efficiencies compared to Cas9 plasmid. This is important because HDR-mediated knockin will only occur in a fraction of DSBs at the target sites. Additionally, Cas9 protein and Cas9 mRNA are DNA-free options so there is no concern of DNA integration resulting from the Cas9 source. Similarly, a Cas9-integrated cell line is not ideal for HDR-mediated knockin because continuous expression of the Cas9 nuclease increases potential for undesired off-targets and mutations.

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