Citation

  • Authors: Zhang K. et al.
  • Year: 2020
  • Journal: Biomaterials 256 120225
  • Applications: in vitro / DNA / PEIpro
  • Cell types:
    1. Name: hNPCs
    2. Name: U-2 OS
      Description: Human bone osteosarcoma
      Known as: U2OS

Method

For gene activation experiments, U2OS cells were seeded onto 24-well plates at a density of 20,000 cells per well. 24 hours after plating, cells were transfected with Cas9-GFP plasmids or a 1:1 mass ratio of dCas9 plasmid:sgRNA plasmids. A total plasmid mass of 0.5 μg per well was transfected using 0.5 μL per well of PEIpro reagent. For synergy experiments, the amount of total sgRNA was kept constant and the mass ratio of each sgRNA was 1:1 or 1:1:1. The cells were harvested 2 d after transfection, and gene expression analysis was performed.

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.

Pubmed