Beyond CRISPR-Cas9, emerging technologies like evoCAST, KNIT and prime assembly are making larger DNA edits possible. But as scientists push the boundaries of genome engineering, can we ensure these breakthroughs are safe, precise and ethical?
By Dona Qiu
Genomic editing technologies have always been an exciting field ever since the late 20th century, with CRISPR Cas9 being a highly notable breakthrough. It was invented by Dr Jennifer Doudna and Dr Emmanuelle Charpentier (The Royal Swedish Academy of Sciences, 2020), and was the first technique to modify specific DNA sequences.
DNA sequences are made of four letters: A, T, C and G; the order in which these letters are arranged carries genetic information. In CRISPR-Cas9, Cas9 is an enzyme acting as a molecular scissor to make a cut in the double-stranded DNA. Guide RNA directs Cas9 to the target region by binding to a complementary sequence on DNA. After the cut, the cell’s own repair mechanism can seal the break. CRISPR-Cas9 was considered revolutionary as it made modifying genomes realistic and provided scientists with insights to design treatments for genetic disorders (Gostimskaya, I, 2022). However, the nature of the cell's self-repairing mechanism is unpredictable and error-prone. During the process of Cas enzyme cutting open both DNA strands, unwanted mutations could be introduced (Ledford, H., (2026), which could insert or delete a DNA letter and change the sequence. ultimately leading to gene inactivation or gene expression, and failing to obtain the desired sequence outcomes (CRISPR Therapeutics, 2025).
Recently, there has been the advent of several genome-editing technologies, which overcome some limitations of CRISPR-Cas9. For example, a Nature article: Bigger than CRISPR? A guide to the latest genome editors (Ledford, H. (2026), introduced evoCAST, which enables insertion of tens of thousands of DNA bases into human cells. Moreover, evoCAST allows scientists to insert DNA sequences at multiple genomic locations relative to potential therapeutic applications, thereby further opening possibilities for treating genetic diseases, and this technique is considered a significant improvement to CRISPR-Cas9.
Another technique is called KNIT. Scientists have tested inserting up to 10,500 base pairs at the site of a single-stranded break using KNIT, and it has surprisingly achieved 90% efficiency (Ledford, H, 2026). All techniques in this article can insert a much higher number of base pairs into DNA, thereby making significant changes to DNA sequences compared to CRISPR-Cas9. What is even more impressive is that all techniques can achieve large DNA insertions without breaking double strands of DNA as CRISPR-Cas9 does. This effectively limits the chance of mutation kicking in while both strands of DNA are broken.
Targeted genomic integration and rearrangement using primer assembly (Levesque, S. et al (2026), introduced the Prime assembly technique, which allows insertion of substantial DNA sequences in both dividing and non-dividing cells, without breaking the double-stranded DNA. Several experiments have been done using this technique, and results have illustrated that prime assembly is capable of supporting extensive genomic rearrangements and transgene integration in human cells. For example, scientists have re-coded a region of DNA associated with the disease dyskeratosis congenita. Its symptoms are bone marrow failure, producing low levels of red blood cells and white blood cells, which are crucial for transporting oxygen and immune response. Primer assembly allows scientists to edit that region of DNA without changing the amino acid sequence encoded by DNA, and has achieved the highest editing efficiency of 33.1%, illustrating that primer assembly not only allows editing DNA sequences on a large scale, but clinical research also shows promising results of successful recoding part of the DNA in human cells; this could be a potential disease-treatment approach to correct DNA sequences in patients.
With the advancement of genome editing, are we ready to rewrite the entire human genome? It has been evidently proven that considerable sequence modification has been made possible by techniques such as KNIT, evoCAST and Primer assembly, some shown potential medical application to recode disease-associated DNA regions to treat genetic disorders. However, scientists must also consider what might happen when an edit goes wrong. For example, off-target editing is still unavoidable, which may trigger undesirable responses in other cells (Aussel, C et al., 2025), so long-term monitoring is needed to ensure long-term safety after editing. The next stage revolution is not simply about learning how to rewrite the human genome, but learning how to do so safely, predictably and fairly, and how reliably those changes improve human lives.
References
Aussel, C., Cathomen, T. and Fuster-García, C. (2025). The hidden risks of CRISPR/cas: Structural variations and genome integrity. Nature Communications, [online] 16(1). doi:10.1038/s41467-025-62606-z.
CRISPR Therapeutics (2025). Gene Editing. [online] CRISPR Therapeutics. Available at: https://crisprtx.com/gene-editing [Accessed 20 Oct. 2026].
Gostimskaya, I. (2022). CRISPR–Cas9: A history of its discovery and ethical considerations of its use in genome editing. Biochemistry (Moscow), [online] 87(8), pp.777–788. doi:10.1134/s0006297922080090.
Ledford, H. (2026). Bigger than CRISPR? A guide to the latest genome editors. Nature, [online] 658(8134), 28 Sept., pp.281–283. doi:10.1038/d41586-026-02964-w.
Levesque, S., Kawashima, N., Hwang, G.-H., Zeng, J., Toskov, V., Barry, T., Mannherz, W., Homfeldt, L., Becerra, B., Schoonenberg, V.A.C., Pinello, L., Agarwal, S. and Bauer, D.E. (2026). Targeted genomic integration and rearrangement using prime assembly. Nature. [online] doi:10.1038/s41586-026-11024-2.
The Royal Swedish Academy of Sciences (2020). The nobel prize in chemistry 2020. [online] NobelPrize.org. Available at: https://www.nobelprize.org/prizes/chemistry/2020/press-release/ [Accessed 20 Oct. 2026].
