Recent applications, future perspectives, and limitations of the CRISPR-Cas system

Abstract

The CRISPR-Cas system has transformed our ability to edit and modify genomes in eukaryotic cells, offering unmatched precision and broad applicability. By utilizing a programmable RNA protein complex to introduce targeted double-strand breaks, the CRISPR-Cas system enables the correction of pathogenic mutations and the modulation of gene function with unprecedented efficiency. Its broad applicability spans the correction of inherited genetic defects through homology-directed repair to the disruption of deleterious alleles via non-homologous end joining. In this review, we first outline the molecular architecture and mechanistic basis of CRISPR-Cas9 and then consider its latest applications in modeling, drug screening, small-molecule-mediated editing, and treating hereditary, autoimmune, and oncological diseases. Emphasis is placed on the generation of disease-relevant cellular and animal models and on the potential of CRISPR-Cas9-mediated gene therapy to address hitherto intractable disorders. Finally, we discuss current challenges including off-target activity, gene editing efficiency, delivery constraints, and immunogenicity and highlight emerging strategies to overcome these hurdles and broaden the clinical impact of CRISPR-Cas systems.

Keywords: CRISPR-Cas9; DNA repair; MT: RNA/DNA Editing; cell engineering; drug screening; gene therapy; genetic editing efficiency.

Graphical abstract

Figure 1

Historical overview of CRISPR technologies CRISPR sequences were first identified in Escherichia coli in 1987 and subsequently observed across various prokaryotes between 1993 and 2005. The discovery of Cas genes in 2002 and the presence of viral DNA fragments within CRISPR spacers in 2005 provided insights into its potential role in adaptive immunity, which was experimentally validated in 2007. In 2013, two independent groups repurposed the CRISPR-Cas9 system as a programmable genome-editing tool. The first clinical application of CRISPR-Cas9 occurred in China in 2016. Over the past 3 years, multiple CRISPR-based therapies have entered clinical stages. In recognition of their pioneering work in developing CRISPR-Cas9 as a genome-editing technology, Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry in 2020.

Figure 2

Schematic overview of the Cas9-gRNA complex and its mechanism of action in the presence of target DNA Compartments of the CRISPR-Cas9 complex bound to the target DNA and guide RNA (gRNA). Cas9 consists of two regions, called the recognition (REC) lobe and the nuclease (NUC) lobe. The REC lobe, comprising the REC1 and REC2 domains, is responsible for nucleic acid recognition. The NUC lobe contains the HNH and RuvC domains and a C-terminal region containing a PAM-interaction domain. The HNH domain and the RuvC domain cleave the target DNA double strand, forming a duplex with crRNA and the other DNA strand, respectively.

Figure 3

The mechanism and outcome of two major DNA repair pathways, NHEJ and HDR, used by CRISPR-Cas9-induced DSBs Cas9 ribonucleoprotein introduces a site-specific DSB, which is primarily repaired by either the error-prone NHEJ pathway or the high-fidelity HDR pathway. In the NHEJ pathway (left), the Ku70/Ku80 heterodimer recognizes and binds to DSB ends, recruiting 53BP1 to protect the ends from resection. DNA-PK stabilizes the complex and promotes synapsis of the DNA ends. XRCC4 recruits DNA ligase IV to ligate the DNA ends, often resulting in insertions or deletions. In the HDR pathway (right), the MRN complex comprising MRE11, RAD50, and NBS1 is recruited to DSBs and facilitates end resection, which is further promoted by CtIP. Single-stranded DNA is coated with RPA, followed by replacement with RAD51 to form a nucleoprotein filament. This filament invades the homologous donor template, forming a D-loop that serves as a platform for DNA synthesis and precise repair. Exo1 and the Dna2/BLM complex contribute to extended end resection, further promoting strand invasion and repair accuracy. For efficient strand invasion and integration, the donor DNA must share homology with the target sequence, and it is often resected at both ends to enable proper alignment and recombination with the host genome.

Figure 4

Applications of the CRISPR-Cas system This diagram illustrates the diverse applications of CRISPR-Cas genome-editing technology across multiple fields. These include (1) functional genomics (e.g., gene knockout and regulatory element studies), (2) disease modeling using animal or cell-based systems, (3) drug target identification and validation, (4) synthetic lethality screening for cancer vulnerabilities, (5) gene therapy and personalized medicine approaches, and (6) agricultural and biotechnology innovations such as crop improvement and livestock engineering.