Statement of Purpose: The CRISPR-Cas9 system is a promising platform for targeted genome editing. A simplified version of this gene-editing tool consists of only two components: the Cas9 nuclease and a single-guide RNA (sgRNA). Cas9 and sgRNA form a nucleoprotein complex that recognizes target DNA locus adjacent to a protospacer adjacent motif (PAM) using the 20-nucleotide portion of the sgRNA. In spite of the progresses made in improving the efficiency and fidelity of this ribonucleoprotein tool, the lack of an efficient delivery system posed a major challenge to therapeutic translation of these bio-macromolecules1. Methods: A bio-inspired DNA nanoclew (NC) based carrier was tailored for the Cas9/sgRNA complex by utilizing the technique of rolling circle amplification2-4. The synthesized DNA NC composed of concatenated single stranded DNA (ssDNA) was designed to be complementary to the targeting portion of sgRNA for efficient Cas9/sgRNA loading. A cationic polymer polyethylenimine was coated onto the Cas9/sgRNA/NC complex to help induce endosome escape (Figure 1). Results: The DNA NC based carrier delivered the Cas9/sgRNA complex into the nucleus of a model cell line that constitutively expressed a destabilized EGFP. Genome editing occurred at the pre-designed genomic locus as determined from the increase of EGFP-negative cells as well as the Surveyor Assay and Sanger Sequencing. Efficient EGFP disruption was also observed in xenograft tumor model in mice that also expressed the EGFP. Conclusions: The DNA NC based delivery system is an efficient and versatile platform for intracellular delivery of the protein/RNA complex, which could be further extended to deliver other DNA associated protein or nucleic acid therapeutics. References: 1. W. Sun, W. Ji, J. M. Hall, Q. Hu, C. Wang, C. L. Beisel and Z. Gu, Angew. Chem. Int. Ed., 2015, DOI: 10.1002/anie.201506030. 2. W. Sun, T. Jiang, Y. Lu, M. Reiff, R. Mo and Z. Gu, J. Am. Chem. Soc., 2014, 136, 14722-14725. 3. W. Sun, Y. Lu and Z. Gu, Therapeutic delivery, 2015, 6, 765-768. 4. W. Sun and Z. Gu, Biomater. Sci., 2015, 3, 1018-1024.

CRISPR–Cas9 delivery by DNA nanoclews for efficient genome editing

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.