Cell penetrating peptide‐mediated nuclear delivery of Cas9 to enhance the utility of CRISPR/Cas genome editing

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