Cell penetrating peptides have long held great potential for delivery of biomolecular cargos for research, therapeutic and diagnostic purposes. They allow rapid, relatively nontoxic passage of a wide variety of biomolecules through the plasma membranes of living cells. However, CPP-based research tools and therapeutics have been stymied by poor efficiency in release from endosomes and a great deal of effort has been made to solve this ‘endosomal escape problem.’ Previously, we showed that use of a reversible, noncovalent coupling between CPP and cargo using calmodulin and a calmodulin binding motif allowed efficient delivery of cargo proteins to the cytoplasm in baby hamster kidney and other mammalian cell lines. The present report demonstrates the efficacy of our CPP-adaptor scheme for efficient delivery of model cargos to the cytoplasm using a variety of CPPs and adaptors. Effective overcoming of the endosomal escape problem is further demonstrated by the delivery of cargo to the nucleus, endoplasmic reticulum and peroxisomes by addition of appropriate subcellular localization signals to the cargos. CPP-adaptors were also used to deliver cargo to myotubes, demonstrating the feasibility of the system as an alternative to transfection for the manipulation of hard-to-transfect cells.

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 InformationThis work was supported by NIH grant R15GM120691