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I'm trying to establish if it's required to add a NLS to Cas9 when expressed (or transfected) in a Eukaryotic cell. Several papers report using a viral NLS, but is it absolutely necessary? Could Cas9 be trafficked to nucleus without addition of a NLS?

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    $\begingroup$ As far as I know it should be needed because it can not enter the nucleus otherwise. $\endgroup$
    – SeRe
    Commented May 9, 2016 at 20:10
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    $\begingroup$ Most of the Cas9 genes you buy for mammalian gene editing experiments will contain at least one copy of the SV40 NLS sequence. $\endgroup$
    – user137
    Commented May 10, 2016 at 0:42

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The transport from cytoplasm to nucleus is mediated by importin family. Importion family recognize NLS and transport proteins such as transcription factors into nucleus. So, NLS is necessary for cas9 to be transported into nucleus.

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    $\begingroup$ can you add some references to your answer? it always helps for others on the site to have further reading and substantiate claims. $\endgroup$ Commented Aug 9, 2016 at 3:22
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Yes, it does.

In general, proteins required in the nucleus must be directed there by some mechanism. https://en.wikipedia.org/wiki/Nuclear_localization_sequence Adding a nuclear localization sequence to the N-term of a protein is a very efficient and established way to accomplish this. Also, as a supporting fact, you can notice that any CRISPR/Cas expression plasmids for mammalian cells on Addgene (here an example http://www.addgene.org/48138/ ) contains one or more NLS at the N-term of the Cas sequence.

Here some links for further reading:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1083765/

https://www.ncbi.nlm.nih.gov/pubmed/27126810

https://www.ncbi.nlm.nih.gov/pubmed/26173234

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