Challenges and Opportunities in the Application of CRISPR-Cas9: A Review on Genomic Editing and Therapeutic Potentials

Abstract

<p>Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) constitute a revolutionary gene-editing technology, allowing precise DNA modifications with vast potential for disease treatment and the creation of genetically modified organisms. This system consists of various components designed to target specific genes, requiring efficient nuclear access within target cells through diverse delivery methods, including physical techniques and carrier-based approaches. Despite its transformative promise, CRISPR faces several challenges, including efficient cellular delivery, off-target effects, immune responses, optimizing editing efficiency, and ethical concerns. Overcoming these hurdles is crucial for fully harnessing its applications. However, CRISPR-Cas9 offers remarkable opportunities for pioneering gene therapies across various disorders, including cancer, and could revolutionize agriculture by engineering disease-resistant crops. A key strength of CRIScPR lies in its adaptability to a broad spectrum of genes, significantly enhancing its versatility. The technology's potential further expands through emerging interdisciplinary integrations, such as artificial intelligence, machine learning, and biological imaging. These advancements can refine CRISPR's precision, improve efficiency, and mitigate existing limitations, positioning it as an indispensable tool in future genetic research. Overall, CRISPR-Cas9 promises transformative progress in healthcare and agriculture, solidifying its role as a cornerstone in the evolution of genetic engineering. </p>.

Keywords: CRISPR-Cas9; Gene editing; Gene therapy; Genetic engineering; Off-target effects.

Fig. 1.

Timeline of significant milestones in the clinical application of CRISPR-Cas9 to human diseases. This infographic highlights the evolution of CRISPR-Cas9 technology, showcasing its journey from initial applications in human cells to successful clinical trials for genetic diseases, emphasizing its transformative role in disease modeling and gene therapy (created with BioRender.com).

Fig. 2.

CRISPR-Cas9 mechanism. The Cas9 protein, guided by an sgRNA, creates a targeted double-strand break (DSB) in genomic DNA near the PAM sequence, unlike RNA interference, which degrades RNA transcripts. sgRNA directs Cas9 for cleavage, leading to DSB repair via two pathways: (1) non-homologous end joining (NHEJ), an error-prone process that may cause insertions or deletions (indels), and (2) homology-directed repair (HDR), which requires a donor DNA template for precise sequence insertion or correction, introduced alongside the CRISPR components (created with BioRender.com).

Fig. 3.

Comparative overview of different Cas protein families used in CRISPR systems. This figure summarizes the size, nuclease activity, molecular targets, and applications of Cas13, Cas9, Cas12, Cas10, and Cas3 proteins. Their differences in target molecules (DNA vs. RNA), cleavage type, and gene size influence their suitability for specific therapeutic, diagnostic, and research purposes (created with BioRender.com).

Fig. 4.

Therapeutic applications of CRISPR-Cas9. The figure categorizes CRISPR-Cas9 applications into three main areas: genetic disorders, cancer therapy, and emerging uses, showcasing CRISPR’s diverse clinical and research impact (created with BioRender.com).