Revolutionizing in vivo therapy with CRISPR/Cas genome editing: breakthroughs, opportunities and challenges

Abstract

Genome editing using the CRISPR/Cas system has revolutionized the field of genetic engineering, offering unprecedented opportunities for therapeutic applications in vivo. Despite the numerous ongoing clinical trials focusing on ex vivo genome editing, recent studies emphasize the therapeutic promise of in vivo gene editing using CRISPR/Cas technology. However, it is worth noting that the complete attainment of the inherent capabilities of in vivo therapy in humans is yet to be accomplished. Before the full realization of in vivo therapeutic potential, it is crucial to achieve enhanced specificity in selectively targeting defective cells while minimizing harm to healthy cells. This review examines emerging studies, focusing on CRISPR/Cas-based pre-clinical and clinical trials for innovative therapeutic approaches for a wide range of diseases. Furthermore, we emphasize targeting cancer-specific sequences target in genes associated with tumors, shedding light on the diverse strategies employed in cancer treatment. We highlight the various challenges associated with in vivo CRISPR/Cas-based cancer therapy and explore their prospective clinical translatability and the strategies employed to overcome these obstacles.

Keywords: CRISPR/Cas; cancer; genome editing; in vivo trials; personalized therapies; precision medicine.

FIGURE 1

Overview of the CRISPR/Cas platform. Classification of the CRISPR/Cas system based on the class, type, and subtype. The number of subtypes within each CRISPR/Cas type is bracketed. For each type, the Cas endonuclease responsible for target cleavage of DNA and/or RNA is denoted (A). Structure of Streptococcus pyogenes Cas9 in complex with guide RNA and target DNA. The protein domains are depicted in crystal (left), schematic (right), and map (bottom) form. Crystal structure was rendered from RCSB PDB ID: 4OO8 (Shin et al., 2017) (B). Mechanism of action of CRIPSR/Cas9. Upon guide RNA-mediated recognition of the PAM sequence, a double-strand break is induced in the target DNA. The DNA damage is subsequently repaired either through error-prone non-homologous end joining (NHEJ) or precise homology-directed repair (HDR) (C).

FIGURE 2

Expanding the potential of the CRISPR/Cas toolkit. Leveraging catalytically dead Cas9 (dCas9) (yellow boxes) and nickase Cas9 (nCas9) (gray boxes) with tailored modulators. This powerful combination allows for a multitude of functionalities, such as precise base editing using adenine base editor (ABE) and cytidine base editor (CBE), prime gene editing, intricate chromatin imaging, and fine-tuned transcription regulation. Additionally, Cas12a (pink boxes) facilitates editing of single-stranded DNA (ssDNA), while Cas13a (green boxes) enables the manipulation of single-stranded RNA (ssRNA). These innovative techniques significantly expand the toolkit’s applications, promising groundbreaking advancements in genetic research and manipulation.

FIGURE 3

Applications of CRISPR/Cas. Overview of some of the most relevant biomedical and research fields for employment of the CRISPR/Cas technology (A). Comparison of in vivo and ex vivo CRISPR/Cas-based therapeutic strategies. In vivo approaches involve the delivery of the CRISPR/Cas system by dint of either a viral or a non-viral vehicle for direct administration into the patient. The HR template denotes the homologous recombination template. By contrast, ex vivo strategies comprise the collection, in vitro editing via CRISPR/Cas, and re-infusion of cells into the patient (B).

FIGURE 4

Approaches for application of CRISPR/Cas in cancer therapy. Strategies for application of the CRISPR/Cas system in cancer therapy based on the identification and targeting of genetic events solely present in malignant cells, eventually causing their specific cell death. These strategies include universal targeting of introns flanking the breakpoint region within a fusion oncogene, patient-specific targeting of mutations present in functional gene regions and the site-specific insertion of suicide genes (SG).