Cell Penetrating Peptide-Mediated Delivery of CRISPR-Cas System

CRISPR-Cas9 allows genome editing in a variety of species. However, many cell types and tissues are resistant to the delivery techniques of the CRISPR-Cas9 complex, limiting its use. The team of David S. Axford from the Kennesaw State University in Georgia introduced a cell-penetrating peptide (CPP) adaptor, TAT-CaM, which allows cytoplasmic delivery and release of a wide variety of biomolecular cargos. The integration of CRISPR-Cas9 using CPP-mediated delivery could make the system much more widely usable.

The team expressed a recombinant Cas9 containing a calmodulin binding site (CBS-Cas9) in Escherichia coli. These TAT-CaM/CBS-Cas9 complexes were then evaluated for their cell penetrating capabilities. After cell penetrating capability and subcellular localization were assessed, a nuclear localization sequence was added to the gene encoding Cas9 to enhance nuclear localization of the Cas9, making it more effective in editing genes.

Due to the success of the complexes, several other CPPs and adaptor proteins and model cargos with subcellular localization signals were also assayed in hopes of developing CPP-adaptor deliveries with altered destinations. Future work could integrate these results into an effective CRISPR-Cas9 delivery and gene editing system.

For more on this study, read the article in FASEB Journal.