Rami M Major, Christine A Mills, Lei Xing, James L Krantz, Justin M Wolter, Mark J Zylka
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Exploring the Cytoplasmic Retention of CRISPR-Cas9 in Eukaryotic Cells: The Role of Nuclear Localization Signals and Ribosomal Interactions.
Cas9 must be localized to the nucleus to access the genome of mammalian cells. For most proteins, adding a single nuclear localization signal (NLS) is sufficient to promote nuclear entry. However, Cas9 nuclear entry appears to be inefficient as multiple NLSs are typically added to Cas9. Here, we found that three different Cas9 variants interact with the ribosome in HEK293T cells, and that this interaction is RNA mediated. Following immunoprecipitation-mass spectrometry of cytoplasmic-localized Cas9-0NLS and nuclear-localized Cas9-4NLS constructs, we identified novel Cas9 interactors in postmitotic neurons, including KEAP1 and additional ribosomal subunits, the latter were enriched in Cas9-0NLS samples. Collectively, our results suggest that Cas9 is sequestered in the cytoplasm of mammalian cells, in part, via interaction with the ribosome. Increasing the number of NLSs on Cas9 and/or increasing the amount of cytoplasmic guide RNA has the potential to outcompete ribosomal RNA binding and promote efficient nuclear localization of CRISPR-Cas9 variants.
CRISPR JournalBiochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍:
In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR.
Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.