Localized delivery of CRISPR/dCas9 via layer-by-layer self-assembling peptide coating on nanofibers for neural tissue engineering

Kunyu Zhang; Wai Hon Chooi; Shuang Liu; Jiah Shin Chin; Aoife Murray; Dean Nizetic; Du Cheng; Sing Yian Chew

doi:10.1016/j.biomaterials.2020.120225

Localized delivery of CRISPR/dCas9 via layer-by-layer self-assembling peptide coating on nanofibers for neural tissue engineering

Biomaterials. 2020 Oct:256:120225. doi: 10.1016/j.biomaterials.2020.120225. Epub 2020 Jul 8.

Authors

Kunyu Zhang¹, Wai Hon Chooi¹, Shuang Liu², Jiah Shin Chin³, Aoife Murray⁴, Dean Nizetic⁵, Du Cheng², Sing Yian Chew⁶

Affiliations

¹ School of Chemical & Biomedical Engineering, Nanyang Technological University, 637459, Singapore.
² Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, School of Materials Science and Engineering, Sun Yat-Sen University, Guangzhou, 510275, China.
³ School of Chemical & Biomedical Engineering, Nanyang Technological University, 637459, Singapore; NTU Institute of Health Technologies, Interdisciplinary Graduate School, Nanyang Technological University, Singapore, 639798, Singapore.
⁴ Lee Kong Chian School of Medicine, Nanyang Technological University, 308232, Singapore.
⁵ Lee Kong Chian School of Medicine, Nanyang Technological University, 308232, Singapore; The Blizard Institute, Barts & The London School of Medicine, Queen Mary University of London, London, E1 2AT, United Kingdom.
⁶ School of Chemical & Biomedical Engineering, Nanyang Technological University, 637459, Singapore; Lee Kong Chian School of Medicine, Nanyang Technological University, 308232, Singapore. Electronic address: sychew@ntu.edu.sg.

PMID: 32738650
DOI: 10.1016/j.biomaterials.2020.120225

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR) systems have a wide variety of applications besides precise genome editing. In particular, the CRISPR/dCas9 system can be used to control specific gene expression by CRISPR activation (CRISPRa) or interference (CRISPRi). However, the safety concerns associated with viral vectors and the possible off-target issues of systemic administration remain huge concerns to be safe delivery methods for CRISPR/Cas9 systems. In this study, a layer-by-layer (LbL) self-assembling peptide (SAP) coating on nanofibers is developed to mediate localized delivery of CRISPR/dCas9 systems. Specifically, an amphiphilic negatively charged SAP^- is first coated onto PCL nanofibers through strong hydrophobic interactions, and the pDNA complexes and positively charged SAP⁺-RGD are then absorbed via electrostatic interactions. The SAPcoated scaffolds facilitate efficient loading and sustained release of the pDNA complexes, while enhancing cell adhesion and proliferation. As a proof of concept, the scaffolds are used to activate GDNF expression in mammalian cells, and the secreted GDNF subsequently promotes neurite outgrowth of rat neurons. These promising results suggest that the LbL self-assembling peptide coated nanofibers can be a new route to establish a bioactive interface, which provides a simple and efficient platform for the delivery of CRISPR/dCas9 systems for regenerative medicine.

Keywords: CRISPR/Cas9; Electrospinning; Gene delivery; Genome editing; Nerve regeneration.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats*
Nanofibers*
Peptides
Rats
Tissue Engineering

Substances

Peptides