Advances in Chitosan-Based CRISPR/Cas9 Delivery Systems

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR) and the associated Cas endonuclease (Cas9) is a cutting-edge genome-editing technology that specifically targets DNA sequences by using short RNA molecules, helping the endonuclease Cas9 in the repairing of genes responsible for genetic diseases. However, the main issue regarding the application of this technique is the development of an efficient CRISPR/Cas9 delivery system. The consensus relies on the use of non-viral delivery systems represented by nanoparticles (NPs). Chitosan is a safe biopolymer widely used in the generation of NPs for several biomedical applications, especially gene delivery. Indeed, it shows several advantages in the context of gene delivery systems, for instance, the presence of positively charged amino groups on its backbone can establish electrostatic interactions with the negatively charged nucleic acid forming stable nanocomplexes. However, its main limitations include poor solubility in physiological pH and limited buffering ability, which can be overcome by functionalising its chemical structure. This review offers a critical analysis of the different approaches for the generation of chitosan-based CRISPR/Cas9 delivery systems and suggestions for future developments.

Keywords: CRISPR/Cas9; cancer; chitosan; chitosan functionalisation; gene delivery system; genetic disorders.

Figure 1

CRISPR/Cas9 mechanism of action. The specific sites on the target sequence (DNA) are recognised by the CRISPR of the single guide RNA. The Cas9 nuclease then cleaves the target sequence, introducing double strand breaks, which are then repaired by homology directed repair or non-homologous end joining.

Figure 2

Chemical structure of chitosan.

Figure 3

Chitosan nanoparticles’ (CsNP) properties conferred by the presence of positive charges of D-glucosamines: these allow CsNPs to complex with the negatively charged nucleic acid, increase mucoadhesion at the intestinal level, boost cellular and nuclear uptake following by endosome escape, and finally, the D-glucosamines allow chemical modifications to enhance chitosan chemical properties.

Figure 4

Graphical representation of the “proton sponge effect” following the uptake of CsNPs by the endosome: (1) protons are transported into the endosome along with chloride and water; (2) endosome swells; (3) endosome bursts releasing the CsNPs; and (4) CsNPs can now reach the nucleus.

Figure 5

Strategies using pristine chitosan backbone. (A) Pristine CsNPs are used to transfect fibroblasts for the treatment of pulmonary arterial hypertension; results showed a decreased mRNA expression of the main gene, BMPR2, involved in the disease. (B) Chitosan is used as coating material of PLGA NPs complexed with CRISPR/Cas9; transfection of HEK-293 cells showed a decreased GFP nuclear expression. (C) Chitosan is used as coating material of alginate NPs complexed with CRISPR/Cas9; following formation of protein corona, treatment of cancer cells showed an enhanced transfection ability due to the plasmid protection from enzymatic degradation.

Figure 6

Strategies improving the specificity of CsNPs. (A) Upon application of a magnetic field, HEK-293 cells were successfully transfected using CRISPR/Cas9 complex immobilised on magnetic peptide-imprinted CsNPs. (B) Conjugation of lactobionic acid to chitosan-induced NPs to preferentially accumulate within ASGPR-overexpressing cells, so as to achieve up to 70% tumour growth inhibition. (C) Two ligands were integrated into CsNPs: AS411 aptamer and hyaluronic acid bind to nucleolin and CD44 receptors overexpressing cancer cells, respectively, so as to achieve up to 90% tumour growth inhibition.

Figure 7

Strategies improving the stability of CsNPs. (A) Chitosan tetrazole; (B) monomethyl ether PEG-chitosan; and (C) chitosan-coated red fluorescent protein (RFP). The transfection efficiency of these strategies was tested using HEK-293 cells ranging between 12.5 and 25%.

Figure 8

Strategies combining higher specificity and stability: (A) trimethyl chitosan modified with DPA and folic acid, (B) carboxymethyl chitosan modified with AS1411 aptamer and biotin, and (C) carboxymethyl chitosan modified with AS1411 aptamer and the transactivating transcriptional activator (TAT).

Figure 9

Carboxymethyl chitosan modified with AS1411 aptamer and the endosomolitic peptide, KALA, as a strategy combining higher specificity, stability, and buffering ability.