Cell penetrating peptide‐mediated nuclear delivery of Cas9 to enhance the utility of CRISPR/Cas genome editing
Abstract
CRISPR/Cas9 is a transformative technology that allows genome editing in a variety of eukaryotic cells. However, many cell types and tissues are resistant to transfection or other delivery of the CRISPR/Cas9 complex, which has limited its utility. Our novel cell‐penetrating peptide (CPP) adaptor, TAT‐CaM, allows cytoplasmic delivery and release of a wide variety of biomolecular cargos. The integration of CRISPR/Cas9 with CPP‐mediated delivery could make CRISPR/Cas9 much more widely utile. Recombinant Cas9 containing a calmodulin binding site (CBS‐Cas9) was expressed in Escherichia coli, from which it was purified using affinity chromatography on a calmodulin sepharose column. TAT‐CaM/CBS‐Cas9 complexes were assayed for cell penetrating capabilities and subcellular localization using confocal microscopy. Several other CPPs and adaptor proteins, e.g. SAP and CALL3, and model cargos with subcellular localization signals were assayed in an attempt to develop CPP‐adaptor deliveries with altered kinetics and destinations. After cell penetrating capability and subcellular localization were assessed, a nuclear localization sequence was added to the gene encoding Cas9. Enhanced nuclear localization of Cas9 would make it more effective in potentially editing genes. Future work will integrate the present results into an effective CRISPR/Cas9 delivery and gene editing system. Support or Funding Information This work was supported by NIH grant R15GM120691
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Axford et al. (2017) demonstrated cellular and sub-cellular localization of CPP-delivered CRISPR/Cas9 RNP using confocal microscopy.
“Axford et al. (2017) in which the authors demonstrated cellular and sub-cellular localization of CPP-delivered CRISPR/Cas9 RNP using confocal microscopy.”
From Delivering CRISPR: a review of the challenges and approaches (2018) · cites this paper as Axford et al., 2017
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“However, most reports are quite recent, such as the work done by Axford et al. (2017) in which the authors demonstrated cellular and sub-cellular localization of CPPdelivered CRISPR/Cas9 RNP using confocal microscopy.”
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“A novel CPP, TAT-calmodulin, was explored to effectively deliver the cargo into the nucleus (Axford et al., 2017).”
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“An adaptor similar to TAT-CaM, consisting of TAT CPP and calmodulin, forms a complex with recombinant Cas9-CBS (calmodulin binding site) RNP, which is efficiently transferred into the cell [259].”
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- Authors
- Axford, D. S., Morris, D. P., & McMurry, J. L.
- Affiliations
- Kennesaw State University (US)
- ORCID
- McMurry, Jonathan L
- Venue
- The FASEB Journal
- Publisher
- Wiley
- Published
- 2017-04
- Volume
- 31
- Issue
- S1
- ISSN
- 0892-6638 (ISSN-L)
- 1530-6860
- OpenAlex
- W2892125362
- Cited by
- 15
- References
- 0
- Citations by year
- 2025: 1 · 2024: 2 · 2023: 1 · 2022: 7 · 2020: 1 · 2019: 2 · 2018: 1
- FWCI
- 0.45
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- The FASEB Journal (publishedVersion)
- Funders
- National Institutes of Health (R15GM120691)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
- Keywords
- cell-penetrating peptides, CRISPR/Cas9, nuclear delivery, calmodulin binding site, Cas9, nuclear localization signal, genome editing, subcellular localization, SAP
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