By Sadie Kim
The Development of CRISPR/Cas9 Gene Editing
In 2012, Jennifer Doudna and Emmanuelle Charpentier published a landmark paper detailing a method for genome editing based on clustered regularly interspaced short palindromic repeats (CRISPR) systems in bacteria. Characterized by distinct, repeating sequences of DNA, CRISPR/CRISPR-associated (Cas) systems are adaptive immune systems found in bacteria and archaea that are capable of recognizing and cleaving the DNA of invading viruses. Doudna and Charpentier elucidated a CRISPR/Cas9 system in which guide RNA composed of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) directs the protein Cas9 to specific DNA sequences complementary to the crRNA-guide sequence, where Cas9 then cleaves the DNA. Substitutions, insertions, and deletions of DNA bases can then occur or be induced at that site as the DNA is repaired. Through designing guide RNAs with sequences complementary to target DNA sequences, the CRISPR/Cas9 system can be programmed to bind to and cut specific sites in the genome. Thus, CRISPR/Cas9 technology enables programmable, site-specific genome editing.[1-3]
CRISPR/Cas9 Therapeutic Applications
The CRISPR/Cas9 system is more accessible to researchers than the previous gene-editing methods of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) due to its simplicity, versatility, and cost-effectiveness.[3] CRISPR/Cas9 technology has the potential to correct genetic mutations underlying various disorders and is thus widely used in the development of treatments for diseases.[3] Also, CRISPR/Cas9 knockout screens, in which CRISPR/Cas9 is used to disrupt various genes, can contribute to the identification and characterization of essential genes in biological processes that may serve as therapeutic targets.[3,4]
Many CRISPR/Cas9 studies have focused on cancer therapy.[2,3] Cancer formation and progression are commonly driven by mutations in oncogenes and tumor suppressor genes.[3] A potential approach in cancer therapeutics is using CRISPR/Cas9 technology to restore the function of TP53, a tumor suppressor gene that is observed to be mutated in approximately 50% of human cancers.[3,5] Also, preclinical studies have demonstrated that using CRISPR/Cas9 to target KRAS, one of the most frequently mutated oncogenes in cancer, can inhibit tumor growth both in laboratory models (in vitro) and living organisms (in vivo).[3,6]
CRISPR/Cas9 technology has also been employed in the treatment of sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT).[2,3] These conditions are among the most prevalent single-gene genetic disorders worldwide, and both involve mutations in the hemoglobin β subunit gene (HBB).[2] High levels of expression of fetal hemoglobin can alleviate the symptoms of SCD and TDT, and fetal hemoglobin is suppressed by the transcription factor BCL11A.[2] A clinical trial developed by CRISPR Therapeutics and Vertex Pharmaceuticals used CRISPR/Cas9 to cleave the BCL11A enhancer region in hematopoietic stem and progenitor cells (HSPCs), which were then transplanted into patients with SCD and TDT. A report published in 2020 found that the first two treated patients in the trial had increased levels of fetal hemoglobin expression over a year after infusion, supporting the potential of CRISPR/Cas9-based therapeutics for treating genetic diseases.[2,7]
Challenges in CRISPR/Cas9 Therapeutics
One of the main challenges to the widespread application of CRISPR/Cas9 technology is reducing off-target effects caused by base mismatches between the guide RNA and non-target DNA sequences. Off-target CRISPR/Cas9 activity can introduce unintended mutations at other sites in the genome during the targeting of a different gene, which poses a major safety concern.[2] To minimize off-target effects, high-fidelity Cas9 mutants, such as HypaCas9 and Cas9-HF1, have been developed.[2,3] These variants demonstrate increased accuracy but may have reduced gene-editing efficiency.[2] Also, increasing the specificity of guide RNAs through the addition of specialized structures can reduce off-target effects.[2]
Another challenge in CRISPR/Cas9-based therapeutics is safely and effectively delivering the gene-editing technology to diseased cells.[2] Methods for CRISPR/Cas9 delivery include viral vectors, lipid nanoparticles (LNPs), polymer nanoparticles (PNPs), biomimetic nanoparticles, and extracellular vesicles (EVs).[2,3] Adeno-associated virus (AAV) vectors are commonly used to deliver gene therapies, but their limited packaging capacity can be a constraint given the large size of many CRISPR/Cas9 systems.[2,3] LNPs are advantageous gene therapy delivery vehicles due to their simple synthesis and stable presence in serum[2], and LNPs encapsulating Cas9 mRNA and guide RNA have been optimized to increase gene-editing efficiency.[3] However, the distribution of CRISPR/Cas9 delivery systems in living organisms (in vivo) must also be taken into account, and the high frequency at which LNPs are enriched in the liver makes them effective delivery vehicles for the treatment of liver diseases but limits their efficiency for treating diseases in other organs.[2] PNPs are also promising CRISPR/Cas9 delivery vehicles because they can be engineered to recognize specific cell membrane surface receptors and release drugs in response to particular tumor microenvironments, such as specific reactive oxygen species (ROS) and pH conditions. The use of PNPs can thus help minimize off-target effects in non-target organs.[2,3]
An additional consideration in the application of CRISPR/Cas9-based therapeutics is using delivery vehicles with low risks of triggering immune responses and other adverse reactions. PNPs and biomimetic nanoparticles can be designed to have low immunogenicity and high biocompatibility.[2,3] Further development of high-fidelity Cas9 variants, guide RNAs with increased specificity, and optimized delivery systems holds promise for the more widespread use of CRISPR/Cas9 technology to treat diseases.[2,3]
References
1. Jinek, Martin, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna, and Emmanuelle Charpentier. “A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity.” Science, June 28, 2012. https://doi.org/10.1126/science.1225829.
2. Li, Tianxiang, Yanyan Yang, Hongzhao Qi, Weigang Cui, Lin Zhang, Xiuxiu Fu, Xiangqin He, Meixin Liu, Pei-feng Li, and Tao Yu. “CRISPR/Cas9 Therapeutics: Progress and Prospects.” Nature News, January 16, 2023. https://doi.org/10.1038/s41392-023-01309-7.
3. Xu, Yangsong, Hao Le, Qinjie Wu, Ning Wang, and Changyang Gong. “Advancements in CRISPR/Cas systems for disease treatment.” Acta Pharmaceutica Sinica B, June 24, 2025. https://doi.org/10.1016/j.apsb.2025.05.007.
4. Dong, Chen, Shuhua Fu, Rowan M. Karvas, Brian Chew, Laura A. Fischer, Xiaoyun Xing, Jessica K. Harrison, et al. “A Genome-Wide CRISPR-Cas9 Knockout Screen Identifies Essential and Growth-Restricting Genes in Human Trophoblast Stem Cells.” Nature News, May 10, 2022. https://doi.org/10.1038/s41467-022-30207-9.
5. Chira, Sergiu, Diana Gulei, Amin Hajito, and Ioana Berindan-Neagoe. “Restoring the P53 ‘Guardian’ Phenotype in P53-Deficient Tumor Cells with CRISPR/Cas9.” Trends in Biotechnology, February 22, 2018. https://doi.org/10.1016/j.tibtech.2018.01.014.
6. Gao, Qianqian, Wenjie Ouyang, Bin Kang, Xu Han, Ying Xiong, Renpeng Ding, Yijian Li, et al. “Selective Targeting of the Oncogenic KRAS G12S Mutant Allele by CRISPR/Cas9 Induces Efficient Tumor Regression.” Theranostics, April 6, 2020. https://pmc.ncbi.nlm.nih.gov/articles/PMC7163449/.
7. Frangoul, Haydar, David Altshuler, M. Domenica Cappellini, Yi-Shan Chen, Jennifer Domm, Brenda K. Eustace, Juergen Foell, and Selim Corbacioglu. “CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia.” The New England Journal of Medicine, December 5, 2020. https://www.nejm.org/doi/10.1056/NEJMoa2031054.