Carrier strategies boost the application of CRISPR/Cas system in gene therapy

Abstract

Emerging clustered regularly interspaced short palindromic repeat/associated protein (CRISPR/Cas) genome editing technology shows great potential in gene therapy. However, proteins and nucleic acids suffer from enzymatic degradation in the physiological environment and low permeability into cells. Exploiting carriers to protect the CRISPR system from degradation, enhance its targeting of specific tissues and cells, and reduce its immunogenicity is essential to stimulate its clinical applications. Here, the authors review the state-of-the-art CRISPR delivery systems and their applications, and describe strategies to improve the safety and efficacy of CRISPR mediated genome editing, categorized by three types of cargo formats, that is, Cas: single-guide RNA ribonucleoprotein, Cas mRNA and single-guide RNA, and Cas plasmid expressing CRISPR/Cas systems. The authors hope this review will help develop safe and efficient nanomaterial-based carriers for CRISPR tools.

Keywords: CRISPR; drug delivery; gene therapy; genome editing; nanomaterials.

FIGURE 1

(A) Key events in the history of CRISPR systems; (B) mechanism of class 2 CRISPR/Cas genome editing systems. (i) Cas9 endonuclease is directed to a specific genomic locus by sgRNA and G‐rich PAM sequence, and then the HNH and RuvC domain of the endonuclease cut the double‐stranded DNA. DNA DSBs will be repaired by NHEJ or HDR (with the donor DNA) mechanisms. (ii) Cas12a (Cpf1) endonuclease is directed to a specific genomic locus by crRNA and a T‐rich PAM sequence (TTTV), and then RuvC‐like domains of the endonuclease cut the double‐stranded DNA. DNA DSBs will be repaired by NHEJ or HDR (with the donor DNA) mechanisms. (iii) Cas13a (C2c2) nuclease is directed to a specific genomic locus by crRNA and protospacer flanking sites (PFS) sequence (3’ A, U, or C (not required by all orthologs)), and then HEPN domains of the endonuclease cut the single‐stranded RNA (ssRNA). (iv) Cas9 nickase with point mutations at HNH or RuvC (not shown above) domain can bind to the target site and cleave a single strand of DNA. DNA single‐strand break (SSBs) will be repaired by base excision repair (BER) or HDR (with the donor ssDNA) mechanisms. (v) The dCas9 variants can bind DNA but cannot cleave it because of the mutation of endonuclease domains, which can be used for gene silencing or activation

FIGURE 2

Overview of the delivery and expression of CRISPR/Cas systems in vivo

FIGURE 3

NLS for Cas9 RNP delivery. (A) Gene editing efficiency is RNP dose dependent. (B) N‐terminal 1–7×NLS‐Cas9‐2×NLS design. (C) Direct delivery of 1–7×NLS‐Cas9‐2×NLS with NPCs led to activation of tdTomato reporter in genome‐edited cells. 4×NLS‐Cas9‐2×NLS designs are more efficient at genome‐editing cells than other designs. (D) Genomic DNA PCR of tdTomato stop locus following RNP direct delivery validates tdTomato+ flow cytometry analysis. Reproduced with permission.^{[ 73 ]} Copyright 2017, Nature Publishing Group

FIGURE 4

Gold NPs for RNP delivery to promote HDR. (A) Schematic illustration of synthesis of CRISPR‐Gold. (B–E) CRISPR‐gold promotes HDR in the dystrophin gene and dystrophin protein expression, and reduces muscle fibrosis in mdx mice, with CTX stimulation. (B) CRISPR‐Gold was injected into the hind leg muscle of eight‐week‐old mdx mice simultaneously with CTX. Bottom: dystrophin mutation sequence and donor DNA design. The donor DNA sequence designed to repair the nonsense mutation is marked in the pink box. The nucleotides marked in green (A, G and G) are silent mutations that prevent Cas9 activity on the edited sequence. (C) CRISPR‐Gold‐induced genome editing in the dystrophin gene was confirmed by deep sequencing. (D) CRISPR‐Gold‐injected muscle of mdx mice showed dystrophin expression (immunofluorescence), whereas control mdx mice did not express dystrophin protein. (E) CRISPR‐Gold reduces muscle fibrosis in mdx mice. Reproduced with permission.^{[ 82 ]} Copyright 2017, Nature Publishing Group

FIGURE 5

(A) Streptococcus pyogenes Cas9 (SpCas9) has a heterogeneous surface charge due to both positive and negative amino acids residues, as well as the negatively charged sgRNA. A schematic illustration for the formation of the covalently crosslinked, yet intracellularly biodegradable, NC for the delivery of the Cas9 RNP complex prepared by in situ free‐radical polymerization. (B) A schematic depiction of the proposed mechanism of the cellular uptake of NCs and the subcellular release of the RNP. Reproduced with permission.^{[ 120 ]} Copyright 2019, Nature Publishing Group

FIGURE 6

(A) Efficacious iPhos lipids were composed of one ionizable amine, one phosphate group and three hydrophobic alkyl tails. On entering acidic endosomes/lysosomes, protonation of the tertiary amine induced a zwitterionic head group, which could readily insert into membranes. (B) Most biological membrane phospholipids possess a zwitterion and adopt a lamellar phase. When iPhos lipids were mixed and inserted into the endosomal membranes, the formed cone shape by small ion pair head and multiple hydrophobic tails enabled hexagonal transformation. (C) Synthetic routes of iPhos: alkylated dioxaphospholane oxide molecules (Pm) were conjugated to amines (nA) to obtain iPhos (nAxPm). ‘x’ in ‘nAxPm’ indicates the number of Pm molecules modified on one amine molecule. (D) A list of 28 amines and 13 alkylated dioxaphospholane oxide molecules used for iPhos synthesis. Reproduced with permission.^{[ 133 ]} Copyright 2021, Nature Publishing Group

FIGURE 7

RBCEVs for Cas9 mRNA/sgRNA delivery. Reproduced with permission.^{[ 136 ]} Copyright 2018, Nature Publishing Group

FIGURE 8

Cationic peptide for Cas9 plasmid delivery. Schematic illustration showing the formation of P‐HNPs and the intracellular activity of Cas9 expression plasmid/sgRNA in performing genome editing or gene activation. Reproduced with permission.^{[ 157 ]} Copyright 2018, National Academy of Sciences

FIGURE 9

Schematic illustration of SPPF‐Dex NPs design and intracellular genome editing process at 808 nm laser irradiation. Reproduced with permission.^{[ 124 ]} Copyright 2019, John Wiley & Sons