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Review
. 2024 Jun 7;12(6):636.
doi: 10.3390/vaccines12060636.

Precision in Action: The Role of Clustered Regularly Interspaced Short Palindromic Repeats/Cas in Gene Therapies

Amrutha Banda  1 , Olivia Impomeni  1 , Aparana Singh  2 , Abdul Rasheed Baloch  3 , Wenhui Hu  3 , Dabbu Kumar Jaijyan  3
Affiliations
Review

Precision in Action: The Role of Clustered Regularly Interspaced Short Palindromic Repeats/Cas in Gene Therapies

Amrutha Banda et al. Vaccines (Basel). .

Abstract

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated enzyme-CAS holds great promise for treating many uncured human diseases and illnesses by precisely correcting harmful point mutations and disrupting disease-causing genes. The recent Food and Drug Association (FDA) approval of the first CRISPR-based gene therapy for sickle cell anemia marks the beginning of a new era in gene editing. However, delivering CRISPR specifically into diseased cells in vivo is a significant challenge and an area of intense research. The identification of new CRISPR/Cas variants, particularly ultra-compact CAS systems with robust gene editing activities, paves the way for the low-capacity delivery vectors to be used in gene therapies. CRISPR/Cas technology has evolved beyond editing DNA to cover a wide spectrum of functionalities, including RNA targeting, disease diagnosis, transcriptional/epigenetic regulation, chromatin imaging, high-throughput screening, and new disease modeling. CRISPR/Cas can be used to engineer B-cells to produce potent antibodies for more effective vaccines and enhance CAR T-cells for the more precise and efficient targeting of tumor cells. However, CRISPR/Cas technology has challenges, including off-target effects, toxicity, immune responses, and inadequate tissue-specific delivery. Overcoming these challenges necessitates the development of a more effective and specific CRISPR/Cas delivery system. This entails strategically utilizing specific gRNAs in conjunction with robust CRISPR/Cas variants to mitigate off-target effects. This review seeks to delve into the intricacies of the CRISPR/Cas mechanism, explore progress in gene therapies, evaluate gene delivery systems, highlight limitations, outline necessary precautions, and scrutinize the ethical considerations associated with its application.

Keywords: CRISPR/Cas; cancer; gene delivery; gene therapy; genetic disease; genome editing; infection; viral vectors.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Mechanism of CRISPR action and gene function study. The CRISPR/Cas9 system creates a double-strand break in the target DNA, which can be repaired using NHEJ (non-homologous end joining) or HDR (homology-directed repair) pathways. NHEJ can result in frameshift mutations, insertion, or deletion of nucleotide bases, while HDR requires a homology donor template to correct mutations precisely and can insert or delete genes at specific locations in the genome. Additionally, Cas9 can be deactivated (dCas9) and fused with other proteins, such as a deaminase, transcription factor, or other proteins, to perform base editing, gene expression, imaging, or epigenome editing. An sgRNA that is about 130–150 nucleotides long guides the Cas effector to the precise genome cleavage site. The sgRNA is created by combining CrRNA and TracrRNA. The spacer within crRNA, which is only 18–21 nucleotides long, is the perfect complement to the target sequence. AD represents adenine deaminases, and CD represents cytidine deaminases. dCas, a modified Cas protein version, can be fused with AD or CD to achieve base editing. When dCas9 is fused with AD, it can convert G-C to T-A, while when fused with CD, A-T to G-C substitution occurs. With this capability, we can potentially correct harmful mutations and cure genetic diseases.
Figure 2
Figure 2
Versatile applications of gene editing with CRISPR/Cas. In the medical field, it has been applied to diagnose diseases and improve CAR T-cell therapy. In the food industry, CRISPR-Cas can enhance crop yields and improve resistance to pests. CRISPR technology has shown great potential in addressing genetic diseases through gene editing. In the field of plant biotechnology, CRISPR-Cas has been used to create crops that are more resistant to environmental stressors and have improved nutritional value. Furthermore, it has been applied to the production of biofuels and the improvement of livestock, resulting in more sustainable practices.
Figure 3
Figure 3
Various delivery methods for CRISPR/Cas. Some of the most promising viral vector-based delivery methods include AAV and LV. Physical methods, like microinjection and electroporation, have also shown great promise. In addition, non-viral vector methods, like nanoparticles, polyplexes, liposomes, and others, have been tested and have shown promising results.
Figure 4
Figure 4
Size comparison of miniature CAS with other CRISPR effectors.
Figure 5
Figure 5
The role of the CRISPR/Cas system in developing vaccine vectors and engineering B-cells to produce new-generation vaccines. (A) CRISPR/Cas can precisely manipulate the genetic material of viruses to create safe and effective vaccines. (B) The CRISPR/Cas system has also been used to engineer B-cells, which are immune cells that produce antibodies. By modifying B-cells using the CRISPR/Cas system, researchers can create new-generation vaccines that are more effective and targeted than traditional vaccines.
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