Perkembangan Teknologi CRISPR dari Cas9 Konvensional Menuju Sistem Editing Presisi Tinggi dalam Aplikasi Bioteknologi Kesehatan

Nur Fajri Nur; Nur Mufliha; Rachmawati Rachmawati

doi:10.31004/riggs.v5i1.7740

Authors

Nur Fajri Nur Universitas Negeri Makassar
Nur Mufliha Universitas Negeri Makassar
Rachmawati Rachmawati Universitas Negeri Makassar

DOI:

https://doi.org/10.31004/riggs.v5i1.7740

Keywords:

CRISPR-Cas9, Genome Editing, Base Editing, Prime Editing, Precision Medicine

Abstract

Perkembangan bioteknologi kesehatan saat ini telah mengalami pergeseran dari pendekatan yang bersifat simptomatik menuju strategi berbasis molekuler yang menargetkan penyebab penyakit pada tingkat genetik. Salah satu terobosan utama dalam bidang ini adalah teknologi CRISPR-Cas9, yang memungkinkan modifikasi DNA secara spesifik, efisien, dan relatif ekonomis. Meskipun demikian, sistem CRISPR-Cas9 konvensional masih menghadapi berbagai keterbatasan, terutama risiko terjadinya double-strand break (DSB) yang dapat memicu mutasi tidak terkontrol serta efek off-target yang tidak diinginkan. Hal ini menekankan perlunya pengembangan teknologi penyuntingan genom yang lebih presisi, aman, dan dapat diterapkan secara klinis. Penelitian ini bertujuan untuk mengkaji evolusi teknologi CRISPR menuju sistem genome editing presisi tinggi dalam konteks bioteknologi kesehatan. Metode yang digunakan adalah Systematic Literature Review (SLR) dengan pedoman PRISMA terhadap 43 artikel yang diperoleh dari basis data Scopus pada periode 2016–2026. Analisis dilakukan secara kualitatif deskriptif dengan membandingkan mekanisme kerja, keunggulan, serta keterbatasan masing-masing generasi teknologi. Hasil kajian menunjukkan bahwa teknologi genome editing telah berkembang dari CRISPR-Cas9 konvensional menuju high-fidelity Cas9, base editing, dan prime editing. High-fidelity Cas9 meningkatkan spesifisitas dengan menurunkan efek off-target, base editing memungkinkan konversi basa tanpa menimbulkan DSB, sedangkan prime editing menawarkan fleksibilitas untuk melakukan substitusi, insersi, dan delesi kecil secara presisi. Evolusi ini tidak hanya meningkatkan keamanan dan akurasi genome editing, tetapi juga memperluas potensi aplikasinya di bidang klinis, khususnya dalam terapi penyakit genetik, pengembangan precision medicine, serta riset bioteknologi lebih lanjut yang menargetkan pengobatan personal dan pencegahan penyakit.

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References

W. Xu, S. Zhang, H. Qin, and K. Yao, “From bench to bedside: cutting-edge applications of base editing and prime editing in precision medicine,” J. Transl. Med., vol. 22, no. 1, p. 1133, Dec. 2024, doi: 10.1186/s12967-024-05957-3.

R. Hermantara, “CRISPR-Cas9: A Story of Discovery, Innovation, and Revolution in Genome Editing,” Indonesian Journal of Life Sciences, pp. 83–104, Mar. 2024, doi: 10.54250/ijls.v6i01.197.

A. S. Sichani, M. Ranjbar, M. Baneshi, F. T. Zadeh, and J. Fallahi, “A Review on Advanced CRISPR-Based Genome-Editing Tools: Base Editing and Prime Editing,” Mol. Biotechnol., vol. 65, no. 6, pp. 849–860, Jun. 2023, doi: 10.1007/s12033-022-00639-1.

M. Chehelgerdi et al., “Comprehensive review of CRISPR-based gene editing: mechanisms, challenges, and applications in cancer therapy,” Mol. Cancer, vol. 23, no. 1, p. 9, Jan. 2024, doi: 10.1186/s12943-023-01925-5.

Z. Liu, D. Guo, D. Wang, J. Zhou, Q. Chen, and J. Lai, “Prime editing: A gene precision editing tool from inception to present,” The FASEB Journal, vol. 38, no. 21, Nov. 2024, doi: 10.1096/fj.202401692R.

M. Ismail and E. Sajjid, “Next-Generation CRISPR Editing: Base Editing, Prime Editing, and the Transition Beyond Double-Strand Breaks,” Pakistan Journal of Medical & Cardiological Review, vol. 5, no. 1, 2026, [Online]. Available: https://pakjmcr.com/index.php/1/about

F. Jiang and J. A. Doudna, “CRISPR–Cas9 Structures and Mechanisms,” Annu. Rev. Biophys., vol. 46, no. 1, pp. 505–529, May 2017, doi: 10.1146/annurev-biophys-062215-010822.

V. Katta et al., “Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease,” Molecular Therapy, vol. 32, no. 10, pp. 3433–3452, Oct. 2024, doi: 10.1016/j.ymthe.2024.07.022.

G. Frati et al., “Safety and efficacy studies of CRISPR-Cas9 treatment of sickle cell disease highlights disease-specific responses,” Molecular Therapy, vol. 32, no. 12, pp. 4337–4352, Dec. 2024, doi: 10.1016/j.ymthe.2024.07.015.

T. Wang et al., “In vivo genome editing with a novel Cj4Cas9,” Commun. Biol., vol. 9, no. 1, p. 152, Dec. 2025, doi: 10.1038/s42003-025-09430-9.

A. A. Abraham and J. F. Tisdale, “Gene therapy for sickle cell disease: moving from the bench to the bedside,” Blood, vol. 138, no. 11, pp. 932–941, Sep. 2021, doi: 10.1182/blood.2019003776.

D. Matsumoto, E. Matsugi, K. Kishi, Y. Inoue, K. Nigorikawa, and W. Nomura, “SpCas9-HF1 enhances accuracy of cell cycle-dependent genome editing by increasing HDR efficiency, and by reducing off-target effects and indel rates,” Mol. Ther. Nucleic Acids, vol. 35, no. 1, p. 102124, Mar. 2024, doi: 10.1016/j.omtn.2024.102124.

P. I. Kulcsár et al., “A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets,” Nat. Commun., vol. 14, no. 1, Dec. 2023, doi: 10.1038/s41467-023-41393-5.

W. Li et al., “Efficient high-precision transgene knock-in by Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 (RED-CRISPR),” Nature Communications , vol. 17, no. 1, Dec. 2026, doi: 10.1038/s41467-025-67239-w.

P. Chen et al., “Highly parallel profiling of the activities and specificities of Cas12a variants in human cells,” Nature Communications , vol. 16, no. 1, Dec. 2025, doi: 10.1038/s41467-025-57150-9.

C. Berthault et al., “Highly Efficient Ex Vivo Correction of COL7A1 through Ribonucleoprotein-Based CRISPR/Cas9 and Homology-Directed Repair to Treat Recessive Dystrophic Epidermolysis Bullosa,” Journal of Investigative Dermatology, vol. 144, no. 6, pp. 1322-1333.e13, Jun. 2024, doi: 10.1016/j.jid.2023.10.035.

K. M. Siow et al., “Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47phox-deficient chronic granulomatous disease,” Mol. Ther. Nucleic Acids, vol. 35, no. 3, Sep. 2024, doi: 10.1016/j.omtn.2024.102229.

A. Eid, S. Alshareef, and M. M. Mahfouz, “CRISPR base editors: Genome editing without double-stranded breaks,” Jun. 11, 2018, Portland Press Ltd. doi: 10.1042/BCJ20170793.

S. N. Begum and S. K. Hasan, “Prime Editing Driven Functional Genomics: Bridging Genotype to Phenotype in the Post-Genomic Era,” Int. J. Mol. Sci., vol. 27, no. 4, p. 1703, Feb. 2026, doi: 10.3390/ijms27041703.

Z. Zhang et al., “Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities,” Mol. Ther. Nucleic Acids, vol. 29, pp. 502–510, Sep. 2022, doi: 10.1016/j.omtn.2022.07.026.

C. Zhong et al., “A Plug-in system for reprogramming the editing patterns of base editors,” Nat. Commun., Dec. 2025, doi: 10.1038/s41467-025-67632-5.

L. Yin, A. Yin, and M. Jones, “Mitochondrial Base Editing of the m.8993T>G Mutation Restores Bioenergetics and Neural Differentiation in Patient iPSCs,” Genes (Basel)., vol. 16, no. 11, Nov. 2025, doi: 10.3390/genes16111298.

D. Leclerc et al., “Gene Editing Corrects In Vitro a G > A GLB1 Transition from a GM1 Gangliosidosis Patient,” CRISPR Journal, vol. 6, no. 1, pp. 17–31, Feb. 2023, doi: 10.1089/crispr.2022.0045.

A. V. Anzalone et al., “Search-and-replace genome editing without double-strand breaks or donor DNA,” Nature, vol. 576, no. 7785, pp. 149–157, Dec. 2019, doi: 10.1038/s41586-019-1711-4.

C. Yang et al., “Prime editor with rational design and AI-driven optimization for reverse editing window and enhanced fidelity,” Nature Communications , vol. 16, no. 1, Dec. 2025, doi: 10.1038/s41467-025-60495-w.

M. Bulcaen et al., “Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells,” Cell Rep. Med., vol. 5, no. 5, May 2024, doi: 10.1016/j.xcrm.2024.101544.

T. Nishiyama et al., “Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy,” Sci. Transl. Med., vol. 14, no. 672, Nov. 2022, doi: 10.1126/scitranslmed.ade1633.

P. J. Chen and D. R. Liu, “Prime editing for precise and highly versatile genome manipulation,” Nat. Rev. Genet., vol. 24, no. 3, pp. 161–177, Mar. 2023, doi: 10.1038/s41576-022-00541-1.

T. Wang et al., “In vivo genome editing with a novel Cj4Cas9,” Commun. Biol., vol. 9, no. 125, p. 1, 2026, doi: 10.1038/s42003-025-09430-9.

N. S. Mikkelsen et al., “Orthogonal CRISPR systems for targeted integration and multiplex base editing enable nonviral engineering of allogeneic CAR-T cells,” Molecular Therapy, vol. 33, no. 12, pp. 6082–6100, Dec. 2025, doi: 10.1016/j.ymthe.2025.08.032.

I. Valdez et al., “A streamlined base editor engineering strategy to reduce bystander editing,” Nature Communications , vol. 16, no. 1, Dec. 2025, doi: 10.1038/s41467-025-63609-6.

D. Wei et al., “AI-guided Cas9 engineering provides an effective strategy to enhance base editing,” Molecular Systems Biology , vol. 21, no. 11, pp. 1563–1580, Nov. 2025, doi: 10.1038/s44320-025-00142-0.

I. P. Joore et al., “Correction of pathogenic mitochondrial DNA in patient-derived disease models using mitochondrial base editors,” PLoS Biol., vol. 23, no. 6, Jun. 2025, doi: 10.1371/journal.pbio.3003207.

J. Geilenkeuser et al., “Engineered nucleocytosolic vehicles for loading of programmable editors,” Cell, vol. 188, no. 10, pp. 2637-2655.e31, May 2025, doi: 10.1016/j.cell.2025.03.015.

Q. Chen et al., “Engineering of Peptide-Inserted Base Editors with Enhanced Accuracy and Security,” Small, vol. 21, no. 14, Apr. 2025, doi: 10.1002/smll.202411583.

K. Xu et al., “Structure-guided discovery of highly efficient cytidine deaminases with sequence-context independence,” Nat. Biomed. Eng., vol. 9, no. 1, pp. 93–108, Jan. 2025, doi: 10.1038/s41551-024-01220-8.

N. Zhao et al., “Evolved cytidine and adenine base editors with high precision and minimized off-target activity by a continuous directed evolution system in mammalian cells,” Nature Communications , vol. 15, no. 1, Dec. 2024, doi: 10.1038/s41467-024-52483-3.

S. Acharya et al., “PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics,” Nat. Commun., vol. 15, no. 1, Dec. 2024, doi: 10.1038/s41467-024-49233-w.

B. Naiisseh et al., “Context base editing for splice correction of IVSI-110 β-thalassemia,” Mol. Ther. Nucleic Acids, vol. 35, no. 2, Jun. 2024, doi: 10.1016/j.omtn.2024.102183.

A. Sharrar et al., “Discovery and engineering of AiEvo2, a novel Cas12a nuclease for human gene editing applications,” Journal of Biological Chemistry, vol. 300, no. 3, Mar. 2024, doi: 10.1016/j.jbc.2024.105685.

Y. Liu et al., “Adenine base editor–mediated splicing remodeling activates noncanonical splice sites,” Journal of Biological Chemistry, vol. 299, no. 12, Dec. 2023, doi: 10.1016/j.jbc.2023.105442.

D. Zhao et al., “Engineered domain-inlaid Nme2Cas9 adenine base editors with increased on-target DNA editing and targeting scope,” BMC Biol., vol. 21, no. 1, Dec. 2023, doi: 10.1186/s12915-023-01754-4.

Y. Qian et al., “A new compact adenine base editor generated through deletion of HNH and REC2 domain of SpCas9,” BMC Biol., vol. 21, no. 1, Dec. 2023, doi: 10.1186/s12915-023-01644-9.

A. T. Joynt et al., “Protospacer modification improves base editing of a canonical splice site variant and recovery of CFTR function in human airway epithelial cells,” Mol. Ther. Nucleic Acids, vol. 33, pp. 335–350, Sep. 2023, doi: 10.1016/j.omtn.2023.06.020.

M. E. Neugebauer et al., “Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity,” Nat. Biotechnol., vol. 41, no. 5, pp. 673–685, May 2023, doi: 10.1038/s41587-022-01533-6.

S. Yin et al., “Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies,” Molecular Therapy, vol. 31, no. 3, pp. 744–759, Mar. 2023, doi: 10.1016/j.ymthe.2022.11.014.

P. I. Kulcsár, A. Tálas, Z. Ligeti, S. L. Krausz, and E. Welker, “SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e,” Nat. Commun., vol. 13, no. 1, Dec. 2022, doi: 10.1038/s41467-022-34527-8.

A. K. Sangree et al., “Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2,” Nat. Commun., vol. 13, no. 1, Dec. 2022, doi: 10.1038/s41467-022-28884-7.

J. R. Davis et al., “Efficient in vivo base editing via single adeno-associated viruses with size-optimized genomes encoding compact adenine base editors,” Nat. Biomed. Eng., vol. 6, no. 11, pp. 1272–1283, Nov. 2022, doi: 10.1038/s41551-022-00911-4.

I. Petković et al., “COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa,” Front. Med. (Lausanne)., vol. 9, Aug. 2022, doi: 10.3389/fmed.2022.976604.

C. Stadelmann, S. Di Francescantonio, A. Marg, S. Müthel, S. Spuler, and H. Escobar, “mRNA-mediated delivery of gene editing tools to human primary muscle stem cells,” Mol. Ther. Nucleic Acids, vol. 28, pp. 47–57, Jun. 2022, doi: 10.1016/j.omtn.2022.02.016.

R. Ibraheim et al., “Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo,” Nat. Commun., vol. 12, no. 1, Dec. 2021, doi: 10.1038/s41467-021-26518-y.

D. Šikrová, V. A. Cadar, Y. Ariyurek, J. F. J. Laros, J. Balog, and S. M. van der Maarel, “Adenine base editing of the DUX4 polyadenylation signal for targeted genetic therapy in facioscapulohumeral muscular dystrophy,” Mol. Ther. Nucleic Acids, vol. 25, pp. 342–354, Sep. 2021, doi: 10.1016/j.omtn.2021.05.020.

S. Balderston et al., “Discrimination of single-point mutations in unamplified genomic DNA via Cas9 immobilized on a graphene field-effect transistor,” Nat. Biomed. Eng., vol. 5, no. 7, pp. 713–725, Jul. 2021, doi: 10.1038/s41551-021-00706-z.

C. A. Vasquez, Q. T. Cowan, and A. C. Komor, “Base Editing in Human Cells to Produce Single-Nucleotide-Variant Clonal Cell Lines,” Curr. Protoc. Mol. Biol., vol. 133, no. 1, Dec. 2020, doi: 10.1002/cpmb.129.

H. Xie et al., “High-fidelity SaCas9 identified by directional screening in human cells,” PLoS Biol., vol. 18, no. 7, Jul. 2020, doi: 10.1371/journal.pbio.3000747.

H. A. Rees et al., “Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery,” Nat. Commun., vol. 8, Jun. 2017, doi: 10.1038/ncomms15790.

A. Edraki et al., “A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing,” Mol. Cell, vol. 73, no. 4, pp. 714-726.e4, Feb. 2019, doi: 10.1016/j.molcel.2018.12.003.