Page 2 - J. Chromatin remodeling and epigenetics

[J. Chromatin remodeling and epigenetics-1]

Epigenetic Regulation Of Gene Structure And Function With

Cell-Permeable Cas9

Yongchan Choi¹˙#, Heeseop Yoo¹, Jinwook Yang¹, Youngsil Choi¹˙*, Daewoong Jo¹˙*

¹RF/Cas9, Cellivery, Seoul 03929, Republic of Korea

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 system is a highly specific
RNA-guided genome modification tool, which is composed of Cas9 nuclease and single stranded guide RNA
(sgRNA). In spite of their great therapeutic potential in treating genetic diseases, however, the clinical applicability

of CRISPR/Cas9 system is limited by low cell/tissue delivery efficiency. To address this issue, cell-permeable (CP)

Cas9 recombinant protein has been developed by fusing sequence-optimized hydrophobic cell-penetrating peptide
(CPP), namely advanced macromolecule transduction domain (aMTD), to deliver Cas9 directly into cells and tissues.
CP-Cas9 showed high cell-permeability and gene-editing activity in T7 endonuclease assay. In addition, pre-

treatment of lipofectamine-coated sgRNA followed by CP-Cas9 showed nuclease activity whereas co-treating CP-
Cas9 with guided RNA did not show any activity, suggesting that an efficient delivery of sgRNA, possibly more by

aMTD conjugation, can be critical for gene-editing activity of Cas9 system. Taken together, this study provides a
novel cell-permeable Cas9 system capable of direct cell delivery, as a therapeutic gene-editing module that is

practical to target human genetic diseases via genome modification.