Recent advances in the delivery and applications of nonviral CRISPR/Cas9 gene editing

Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed.

Keywords: CRISPR/Cas9; Clinical applications; Gene therapy; Gene-editing; Nonviral delivery system; Vectors.

Fig. 1

Overview of the Cas9 endonuclease. TracrRNA is attached to 3′ end of crRNA and acts an anchor to the Cas9 endonuclease, this combined RNA molecule is known as a single guide RNA (sgRNA). Cas9 scans potential target DNA for the appropriate protospacer adjacent motif (PAM). When the protein finds the PAM, a confirmation change occurs leading to unwinding of the DNA allowing for interaction between the crRNA and DNA. The RuvC and HNH domains then cut the target DNA if complementary binding occurs between the guide and seed region

Fig. 2

Delivery of CRISPR/Cas9 in the form of pDNA, mRNA, and RNP. pDNA will converge into mRNA pathway, and mRNA will converge into the RNP pathway. pDNA steps: Successful delivery of pDNA results in the plasmid being transported to the nucleus, where transcription can occur. Transcribed sgRNA and Cas9 mRNA is exported from the nucleus to the cytoplasm. mRNA steps: mRNA is translated to yield the folded Cas9 endonuclease, which then interacts with sgRNA to form the Cas9 RNP complex. Cas9 RNP steps: the RNP complex is imported into the nucleus, where genome editing occurs

Fig. 3

Summary of methods for targeted CRISPR/Cas9 delivery. A Cargo loading shows different forms of the CRISPR/Cas9 system which can be loading into non-targeted delivery vectors, with the examples being liposomes, polymer nanoparticles, and peptide. B Modifications for targeting is achieved through attaching with a targeting moity (such as a peptide, antibody, or aptamer) that can selectively bind to a receptor/cell surface protein/sugar and allow for active targeted cellular delivery. Modification with tissue specific promoter can direct a delivery system to a cell of interest. C Passive targeting can be divided into tumor and organ targeting. Tumor targeting occurs via the use of confirmational changes of the delivery systems under acidic environment of tumor micro-environment. Organ targeting uses a lipid nanoparticle approach in using the biophysical (siz, and charge) properties of the formulations resulting in accumulation in a specific organ, e.g., liver, lung, and spleen depending on formulation used, leading to targeted delivery