Beyond CRISPR/Cas9: Understand, Choose, and Use the Right Genome Editing Tool

CRISPR/Cas9 sparked the gene editing revolution for both research and therapeutics.¹ But this system still showed severe issues that limited its applications. The most prominent were the heavy reliance on PAM sequences, delivery limitations, double-stranded breaks that prompt unintended edits and cell death, and editing inefficiency (both in targeting and in knock-in reliability).

Despite this, “CRISPR helped turn genome editing from a specialized technique into a standard research tool. We now have clinical evidence that CRISPR-based approaches can change the course of disease,” said Mollie Schubert, Senior Innovation Product Manager at Integrated DNA Technologies.

“Since the first CRISPR/Cas9 applications, advances in editing technologies—including base editors, prime editors, and recombinases—have expanded the precision and control of genome modifications. Continued improvements in delivery platforms… have and will continue to broaden the range of therapeutically addressable targets,” added Loren Schoch, VP Gene Editing Therapeutics at Aldevron.

But with this new wave of next-generation gene editing techniques, tools, and reagents, scientists need to make the right decision for their experiments, whether they are for research or future therapies.

This article covers the current approaches and key considerations to help you choose the right one.




Figure 1. New technologies in genome editing. The image shows some of the emerging methods that are driving genome editing forward. Source: Pacesa, Pelea & Jinek. Cell, 2024. Figure 5, under CC_BY 4.0.²

A brief look at current gene editing approaches (2026)

The first part of choosing the right approach is to understand them. This brief table showcases the main general approaches of gene editing and how they work.

Table 1. Main gene editing approaches and how they work



Read more about the evolution of gene editing approaches and how they work.

Main considerations when choosing between gene editing approaches

With such a wide palette of techniques, and advances being published constantly, decisions can be hard to make. Using the wrong approach will cost researchers money and time.

“A common mistake is optimizing only for on-target efficiency without thinking through the broader biological consequences of the editing approach. Researchers should start with the biological question: what change is needed, in which cell type, and how durable does that change need to be?” Schubert stated.

When it comes to therapies, some of these questions are of extreme importance. “Delivery remains a primary hurdle, as many cell and tissue types are inherently difficult to access or are resistant to uptake of genome editing payloads. Scalable manufacturing, regulatory clarity, and, ultimately, reimbursement remain important barriers to broader adoption. Continued innovation in these areas will help improve the predictability, accessibility, and scalability of future treatments,” Schoch added.

It is important to remember that despite the significant advances, gene editing is still unpredictable and carries risk. For example, base editing and prime editing still show multiple off-target effects,^8,9 and gene therapy trials have resulted in holds, side effects, and deaths because of issues with the delivery methods and editing molecules.¹⁰

Preventing the main issues

Once a question is asked and an experimental approach chosen, researchers can and should still try to ensure the best results.

“The biggest sources of variability (in a gene editing experiment) are delivery efficiency, cell type and cell state, repair pathway activity, and reagent quality. Researchers can control for these by validating delivery up front, using the right controls and orthogonal readouts, choosing formats suited to the cell model, and sourcing consistent, high-quality editing components,” explained Schubert.

Here is how you can apply those tips directly to your experiments:

1. Validate delivery up front using a reporter with your delivery system (such as a fluorescent protein) to see how many cells actually receive the payload.
2. Perform orthogonal readouts by using two or more independent methods to assess the results of the experiments and determine if the edits occurred.
3. Choose the right format for your cell model, as plasmid DNA, mRNA, or viral vectors do not work the same way on different cell types.
4. Source high-quality editing components, making sure RNAs and proteins have high purity, low lot-to-lot variability, and come with thorough QC.

From research to therapeutics

Advancing gene editing from research settings to clinical trials and new therapies faces multiple challenges. Among them, regulatory hurdles, manufacturing, and reimbursement matter. But on the scientific side, off-target edits and delivery are the main safety issues for patients and researchers.

“Before advancing a gene-editing therapy, off-target characterization needs to be comprehensive, product-specific, and tied to the intended clinical context. That usually means combining in silico prediction, unbiased genome-wide methods, targeted deep sequencing, and assessment of chromosomal integrity in relevant cells or tissues, with enough sensitivity to support a clear risk-benefit rationale,” Schubert explained.

“Comprehensive evaluation of editing specificity and genomic integrity is now a core component of therapy development from the earliest stages and safety profiling before translating into the clinic,” Schoch added.

Delivery is crucial because approaches from viral vectors to lipid nanoparticles determine which cells, and how many, receive the treatment. Delivery often results in side effects, with adverse reactions caused by the delivery format as much as by the molecular payloads¹¹.

What the next decade of genome editing looks like

Genome editing will continue to evolve. From modified proteins and RNAs to better manufacturing and delivery approaches, changes will keep expanding what researchers can do both in the lab and the clinic. And this field has the potential to revolutionize not only research and human health, but also agriculture, animal health, and the environment.²

The experts working on gene editing seem to agree that new innovation will continue to advance the field. “In 10 years, genome editing is likely to be an established therapeutic modality. Together, advances in delivery, translational experience, and regulatory clarity will drive genome editing from a promising innovation to a routine component of the therapeutic toolkit,” Schoch explained. And if innovation continues at the same pace as it has since CRISPR-Cas9 was first demonstrated as a programmable genome-editing tool in 2012, Schoch is likely to be more right than wrong in his prediction.

References

1. Gostimskaya I. CRISPR–Cas9: A History of Its Discovery and Ethical Considerations of Its Use in Genome Editing, 2022, Biochemistry (Moscow), DOI:10.1134/S0006297922080090
2. Pacesa M, Pelea O, Jinek M. Past, present, and future of CRISPR genome editing technologies, 2024, Cell, DOI:10.1016/j.cell.2024.01.042
3. Guan K, Fregoso Ocampo R, et al. Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency, 2026, Nature Structural & Molecular Biology, DOI:10.1038/s41594-026-01788-6
4. Kantor A, McClements ME, MacLaren RE. CRISPR-Cas9 DNA Base-Editing and Prime-Editing, 2020, International Journal of Molecular Sciences, DOI:10.3390/ijms21176240
5. Rainaldi J, Mali P, Nourreddine S. Emerging clinical applications of ADAR based RNA editing, 2025, Stem Cells Translational Medicine, DOI:10.1093/stcltm/szaf016
6. Witte IP, Lampe GD, et al. Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase, 2025, Science, DOI:10.1126/science.adt5199
7. Otero CP, Qi LS. Rewriting the epigenome: CRISPR tools for biological discovery and therapeutics, 2026, Current Opinion in Biomedical Engineering, DOI:10.1016/j.cobme.2026.100658
8. Shmuel-Eidelman M, Cohen-Fultheim R, Eisenberg E, Levanon EY. Off-target RNA editing hotspots caused by base editors, 2026, Molecular Therapy, DOI:10.1016/j.ymthe.2025.12.043
9. Zheng J, Wu M, et al. Prime Editing Exhibits Limited Genome-Wide Off-Target Effects in Cellular and Embryonic Gene Editing, 2026, Cells, DOI:10.3390/cells15050438
10. Wills CA, Drago D, Pietrusko RG. Clinical holds for cell and gene therapy trials: Risks, impact, and lessons learned, 2023, Molecular Therapy — Methods & Clinical Development, DOI:10.1016/j.omtm.2023.101125
11. Shchaslyvyi AY, Antonenko SV, Tesliuk MG, Telegeev GD. Current State of Human Gene Therapy: Approved Products and Vectors, 2023, Pharmaceuticals (Basel), DOI:10.3390/ph16101416

About the author: Darío Sánchez Martín is a scientific writer and molecular biologist, specializing in biotechnology, molecular biology, and nanotechnology. He holds a Ph.D. in Biotechnology from Uppsala University, where he developed nanoparticle-based visual and magnetic assays to detect antimicrobial resistance genes. He co-founded and works at Helixa Communications as a scientific writer, helping life-science companies turn complex science into clear and persuasive content for diverse audiences.