Advancing Miniature Gene Editing: High-Efficiency IscB Variants Enable In Vivo Therapeutic Potential

Over the past decade, gene editing technologies, particularly CRISPR/Cas9 and its derivatives, have rapidly advanced, enabling more precise and efficient genetic modifications. This progress offers new possibilities for better understanding life processes and developing precise gene therapies. The first ex vivo gene editing therapy based on CRISPR/Cas9, Casgevy, was approved at the end of 2023 for the treatment of thalassemia and sickle cell anemia1. In vivo gene therapies, delivered via liver-targeted LNP-mRNA, have also shown promising clinical trial data. Gene editing has thus entered the era of clinical applications. However, there are still significant challenges in targeting organs other than the liver for in vivo gene editing. Currently, the main vectors for gene delivery beyond the liver are adeno-associated viruses (AAVs), but their packaging limit of about 4.7 kb makes it difficult to deliver nucleases like Cas9 and its derivatives. Therefore, finding small, efficient gene editing systems is crucial for safe and effective in vivo delivery.

In recent years, several small Cas proteins have been reported, including the miniature Cas12f proteins2-4, their evolutionary ancestor TnpB5, 6, and eukaryotic homologs Fanzor7, 8. However, because the Cas12f family has only one RuvC domain, it is challenging to use them for derived technologies like base editing and prime editing, which rely on nickase enzymes.

In 2021, Feng Zhang’s team discovered the IscB nuclease, encoded by the IS200/IS605 transposon superfamily, through targeted mining and analysis of sequencing data9. IscB, a possible evolutionary ancestor of Cas9, generally has only about 400-500 amino acids (approximately one-third the length of SpCas9) and uses a non-coding RNA (ωRNA) to guide the protein to recognize and cut DNA. This makes IscB a potential candidate for creating a nickase enzyme that can be fused with cytidine deaminase (APOBEC), adenosine deaminase (TadA), or reverse transcriptase (RT) to construct miniaturized base editors (BE) or prime editors (PE) that can be fully packaged into a single AAV, offering significant clinical application potential. However, IscB’s activity in mammalian cells is very limited. For example, the editing efficiency of OgeuIscB in HEK293FT cells is less than 5%. Thus, enhancing IscB’s gene editing activity to levels comparable to Cas9 is a primary challenge.

On August 2, 2024, Dali Li’s team from East China Normal University published a study in Molecular Cell titled “Engineering IscB to Develop Highly Efficient Miniature Editing Tools in Mammalian Cells and Embryos.” The research combines protein engineering, RNA structure optimization, and embryo injection technologies to successfully obtain IscB variants (eIscB-D) with ultra-high editing activity in human and mouse-derived cell lines. By fusing IscB nickase (eIscBH339A) with adenosine deaminase (TadA-8e) or cytidine deaminase (hA3A*), they developed highly active miniature single-base editors eiABE and eiCBE. This system showed efficient in vivo editing activity in mouse embryos and can be used to construct animal models of diseases such as albinism.

To enhance IscB protein activity, researchers introduced arginine mutations at key positions based on rational design10, 11. After three rounds of iterative screening, they obtained an enhanced IscB (named eIscB) with editing efficiency up to 22.4 times higher than wild-type IscB, with an average improvement of 7.5 times. Additionally, by fusing a non-sequence-specific DNA double-strand binding protein (HMG-D), they increased IscB’s affinity for target DNA, achieving a maximum editing efficiency of 91.3% with high-activity eIscB (eIscB-D). They further optimized the guide RNA, obtaining a highly efficient ωRNA (named eωRNA), which is about 20% shorter than the wild-type ωRNA, reducing the difficulty of industrial synthesis. The optimized eIscB-D/eωRNA system achieved an average editing efficiency improvement of 20.2 times compared to the original IscB/ωRNA.

By introducing alanine mutations at key catalytic sites of the RuvC domain and screening, the researchers developed IscB nickase (eIscBH339A), and fused it with adenosine deaminase (TadA-8e) and cytidine deaminase (hA3A*) to create highly active miniature single-base editors eiABE and eiCBE, with maximum editing efficiencies of 73.6% and 79.2%, respectively.

Previously, there was no evidence that IscB could produce efficient editing in mice. The researchers first screened targets for the PCSK9 and Tyr genes in the mouse N2a cell line. Sequencing results showed that eIscB-D achieved 58% editing efficiency at the PCSK9-sg29 target and 47.1% at the Tyr-sg21 target. They then injected the eIscB-D/eωRNA system targeting exon 1 of the Tyr gene into mouse embryos, disrupting the expression of the albinism gene, and successfully created a mouse model of albinism. In the F0 generation, 75% (9/12) of the mutant mice showed high-efficiency editing (average editing efficiency of 58.8%), with 5 mice exhibiting nearly 100% editing, resulting in a completely albino phenotype. This study demonstrated for the first time that eIscB-D can produce efficient editing in mouse-derived cell lines and can efficiently create disease animal models through embryo injection.

In summary, this study successfully developed highly active miniature gene editing tools based on IscB, achieving efficient in vivo editing in mice for the first time. This research significantly increases the potential for safe and efficient delivery of base editing or prime editing systems using a single AAV vector, expands the application scenarios of gene editing tools, and provides efficient candidate technologies for future in vivo gene therapy.

Reference:
1. Wong, C. (2023). UK first to approve CRISPR treatment for diseases: what you need to know. Nature 623, 676–677.
2. Pausch, P., Al-Shayeb, B., Bisom-Rapp, E., Tsuchida, C.A., Li, Z., Cress, B.F., Knott, G.J., Jacobsen, S.E., Banfield, J.F., and Doudna, J.A. (2020). CRISPR-CasΦ from huge phages is a hypercompact genome editor. Science 369, 333-337.
3. Harrington, L.B., Burstein, D., Chen, J.S., Paez-Espino, D., Ma, E., Witte, I.P., Cofsky, J.C., Kyrpides, N.C., Banfield, J.F., and Doudna, J.A. (2018). Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science 362, 839-842.
4. Chen, W., Ma, J., Wu, Z., Wang, Z., Zhang, H., Fu, W., Pan, D., Shi, J., and Ji, Q. (2023). Cas12n nucleases, early evolutionary intermediates of type V CRISPR, comprise a distinct family of miniature genome editors. Mol. Cell 83, 2768-2780.e2766.
5. Karvelis, T., Druteika, G., Bigelyte, G., Budre, K., Zedaveinyte, R., Silanskas, A., Kazlauskas, D., Venclovas, Č., and Siksnys, V. (2021). Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease. Nature 599, 692-696.
6. Xiang, G., Li, Y., Sun, J., Huo, Y., Cao, S., Cao, Y., Guo, Y., Yang, L., Cai, Y., Zhang, Y.E., et al. (2023). Evolutionary mining and functional characterization of TnpB nucleases identify efficient miniature genome editors. Nat. Biotechnol. 42, 745-757.
7. Saito, M., Xu, P., Faure, G., Maguire, S., Kannan, S., Altae-Tran, H., Vo, S., Desimone, A., Macrae, R.K., and Zhang, F. (2023). Fanzor is a eukaryotic programmable RNA-guided endonuclease. Nature 620, 660-668.
8. Jiang, K., Lim, J., Sgrizzi, S., Trinh, M., Kayabolen, A., Yutin, N., Bao, W., Kato, K., Koonin, E.V., Gootenberg, J.S., et al. (2023). Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses. Sci. Adv. 9, eadk0171.
9. Altae-Tran, H., Kannan, S., Demircioglu, F.E., Oshiro, R., Nety, S.P., McKay, L.J., Dlakić, M., Inskeep, W.P., Makarova, K.S., Macrae, R.K., et al. (2021). The widespread IS200/605 transposon family encodes diverse programmable RNA-guided endonucleases. Science 374, 57-65.
10. Xu, X., Chemparathy, A., Zeng, L., Kempton, H.R., Shang, S., Nakamura, M., and Qi, L.S. (2021). Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing. Mol. Cell 81, 4333-4345.e4334.
11. McGaw, C., Garrity, A.J., Munoz, G.Z., Haswell, J.R., Sengupta, S., Keston-Smith, E., Hunnewell, P., Ornstein, A., Bose, M., Wessells, Q., et al. (2022). Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing. Nat. Commun. 13, 2833.https://doi.org/10.1016/j.molcel.2024.07.007

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