CRISPR 101: Any Base Transversion Editors

In our last post, we talked about the first base transversion editors: CGBEs, or C → G Base Editors. CGBEs first convert a cytosine (C) to uracil (U), just like Cytosine Base Editors (CBEs). But unlike CBEs, CGBEs then excise the U to create an abasic (empty) DNA site using either fused or endogenous Uracil DNA N-glycosylase (UNG) enzymes. Various other proteins included in different CGBE tools allow the researcher to direct the repair of the abasic site.

But what if an editor could excise other bases? Could other transversion editors be close at hand?

Once again, this post has a lot of acronyms, so we've included a glossary at the end of this post!

Figure 1: Base transitions are edits between the purine bases (adenine, guanine, and hypoxanthine) or between the pyrimidine bases (cytosine, thymine, and uracil). Base transversions are edits that convert a purine to a pyrimidine or vice-versa. Note: hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com.

Inosine excision leads to the first adenine transversion editors

Uracil excision is the most common type of base excision repair. But mammals also have an enzyme to remove inosine from DNA: N-methylpurine DNA glycosylase (MPG), also known as alkyladenine DNA glycosylase (AAG). When adenine base editors (ABEs) were originally being developed for A → G edits, researchers tried inhibiting MPG/AAG to improve editing efficiency, but it didn’t help: it seemed that the endogenous enzyme is not efficient enough to hinder adenine base editing.

Once base transversion editors were developed, though, researchers at HuidaGene Therapeutics guessed that fusing MPG to an ABE would allow it to act on newly-deaminated inosines.

Pro tip! The nucleoside inosine consists of the nucleobase hypoxanthine + a ribose sugar.

Their fusion of wild-type MPG to an ABE was able to edit A → Y (Y = T or C) in 67.17% of screening cells (Tong, Wang, et al., 2023). Based on this promising start, the team used rational mutagenesis to produce the adenine transversion editor AYBEv3. This tool still retained considerable A → G editing activity at most sites, in addition to A → C and A → T activity. To drive selective A → T editing, they co-expressed Polη, a translesion synthesis polymerase known to preferentially incorporate A bases across from abasic sites. This approach resulted in a maximum A → T product purity of 66%.

Pro tip! Abasic sites are also called apurinic or apyrimidinic sites and abbreviated as AP sites.

A collaborative team from the David Liu and Dali Li labs reported a similar tool shortly thereafter, using mouse instead of human AAG (remember, MPG and AAG are the same protein!) (Chen et al., 2024). They called their initial editor AXBE, for A → X base editor (X = any nucleotide). To create a more specific A → C base editor (ACBE), they inserted the deaminase into the C-terminal domain of Cas9, which narrowed the editing window and reduced A → G bystander edits. Their final tool, ACBE-Q (for an N108Q mutation in the deaminase), produced an average A → C product purity of 48%.

Like CGBEs, these adenine transversion editors produce a lot of indels (up to 40% at one site tested with AYBEv3!). Although AYBE, AXBE, and ACBE do not approach the high product purity of CBEs and ABEs, they could be useful for saturation mutagenesis, and they provide a strong proof of concept for further development.

Figure 2: Mechanisms of deaminase-based adenine transversion editors. Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate natural cellular processes. Created with BioRender.com.

The first deaminase-free base editor allows guanine transversions

And further development was on its way! All base editors up to this point relied on deaminase enzymes, either to directly accomplish the base edit or to create the substrate for a base excision repair pathway. The team behind AYBEv3 introduced the first deaminase-free base editor when they realized that MPG could be engineered to act directly on unmodified G bases, resulting in a G → Y (Y = T or C) base editor, which they called glycosylase-based guanine base editor (gGBE) (Tong, Liu, et al., 2023).

gGBE consists only of engineered MPG fused to nCas9. First, MPG removes the G base from DNA to create an abasic site. Like in most other base editor designs, the Cas9 nicks the non-edited strand to encourage the cell to repair the DNA using the edited strand as a template — in this case, encouraging it to incorporate a new base across from the abasic site before filling the empty space. Over several rounds of rational mutagenesis, the team progressed to gGBEv6.3, which was capable of 80% efficient G → Y editing. Like other transversion editors, this editor had relatively high indel rates ranging from 4.7% to 30% at tested sites. Still, it opened a new avenue for base editor development going forward.

Figure 3: Mechanism of gGBE. Created with BioRender.com.

Base excision editing extends to thymine

Since an enzyme that excises inosine can be modified to target guanosine instead, perhaps analogous engineering of UNG, which excises uracil, could allow it to recognize thymine (T) or cytosine (C). Several different teams succeeded with this approach around the same time (He et al., 2024; Tong et al., 2024; Ye et al., 2024; Yi et al., 2024). In each case, a UNG enzyme was modified to recognize T and/or C substrates. The HuidaGene Therapeutics team has deposited their editors, called gTBE and gCBE, with Addgene. gTBE can introduce T → C and T → G edits, though with limited selectivity between those outcomes, with an average efficiency of 39.3%. gCBE is a deaminase-free C → G editor with a different editing window than existing CGBEs. This distinct approach means researchers can now select the best tool for their application from among many options. 

Conclusion

With these advances, every base can now be directly targeted by a base editor, although the efficiency, purity, and indel rate of each tool vary considerably. And there’s so much more exciting protein engineering going on in the base editing field! From CRISPR-free approaches to mitochondrial base editing to base editors optimized for particular species and more, base editors are getting smaller, more accurate, more efficient, and more diverse. We can’t wait to see what tools the field will come up with next!

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References and Resources

Glossary

Here’s a quick refresher if you ever lose track of which acronym is which.

Acronym	Full name	Notes
	Base transition	Base change between purines (A ↔ G) or between pyrimidines (C ↔ T).
	Base transversion	Base change from purine (A or G) to pyrimidine (C or T) or vice-versa.
CBE	Cytosine Base Editor	Converts C → T; the first type of base editor invented.
ABE	Adenine Base Editor	Converts A → G; the second type of base editor invented.
CGBE	C → G Base Editor	Converts C → G by fusing a uracil DNA glycosylase to a CBE; the first type of base transition editor invented.
	Abasic site	A DNA site with the nucleobase removed. Also called an apurinic / apyrimidinic (AP) site.
UNG / UDG	Uracil DNA N-Glycosylase	Removes uracil from DNA, creating an abasic site.
MPG / AAG	N-methylpurine DNA glycosylase / alkyladenine DNA glycosylase	Removes hypoxanthine from DNA, creating an abasic site. The same protein (or its homologs) has more than one name.
Indel	Insertion/deletion	A type of unwanted DNA edit.
AYBE, AXBE, ACBE	Adenine transversion editors	Converts A to Y (T or C), X (any base), or C, respectively.
gGBE, gTBE, gCBE	Glycosylase-based G, T, or C base editors	Excises G, T, or C, respectively, to be replaced with another base.

References

Chen, L., Hong, M., Luan, C., Gao, H., Ru, G., Guo, X., Zhang, D., Zhang, S., Li, C., Wu, J., Randolph, P. B., Sousa, A. A., Qu, C., Zhu, Y., Guan, Y., Wang, L., Liu, M., Feng, B., Song, G., … Li, D. (2024). Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos. Nature Biotechnology, 42(4), 638–650. https://doi.org/10.1038/s41587-023-01821-9

He, Y., Zhou, X., Chang, C., Chen, G., Liu, W., Li, G., Fan, X., Sun, M., Miao, C., Huang, Q., Ma, Y., Yuan, F., & Chang, X. (2024). Protein language models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing. Molecular Cell, 84(7), 1257-1270.e6. https://doi.org/10.1016/j.molcel.2024.01.021

Tong, H., Liu, N., Wei, Y., Zhou, Y., Li, Y., Wu, D., Jin, M., Cui, S., Li, H., Li, G., Zhou, J., Yuan, Y., Zhang, H., Shi, L., Yao, X., & Yang, H. (2023). Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase. National Science Review, 10(8), nwad143. https://doi.org/10.1093/nsr/nwad143

Tong, H., Wang, H., Wang, X., Liu, N., Li, G., Wu, D., Li, Y., Jin, M., Li, H., Wei, Y., Li, T., Yuan, Y., Shi, L., Yao, X., Zhou, Y., & Yang, H. (2024). Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467-024-49343-5

Tong, H., Wang, X., Liu, Y., Liu, N., Li, Y., Luo, J., Ma, Q., Wu, D., Li, J., Xu, C., & Yang, H. (2023). Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase. Nature Biotechnology, 41(8), 1080–1084. https://doi.org/10.1038/s41587-022-01595-6

Ye, L., Zhao, D., Li, J., Wang, Y., Li, B., Yang, Y., Hou, X., Wang, H., Wei, Z., Liu, X., Li, Y., Li, S., Liu, Y., Zhang, X., & Bi, C. (2024). Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells. Nature Biotechnology, 42(10), 1538–1547. https://doi.org/10.1038/s41587-023-02050-w

Yi, Z., Zhang, X., Wei, X., Li, J., Ren, J., Zhang, X., Zhang, Y., Tang, H., Chang, X., Yu, Y., & Wei, W. (2024). Programmable DNA pyrimidine base editing via engineered uracil-DNA glycosylase. Nature Communications, 15(1), 6397. https://doi.org/10.1038/s41467-024-50012-w

Additional Resources on the Addgene Blog

• CRISPR 101: Cytosine and Adenine Base Editors
• CRISPR 101: Cytosine Transversion Editors
• Read about four base editor reporters

Resources on Addgene.org

• Browse CRISPR plasmids for base editing
• Get a broad overview with our CRISPR Guide
• Check out our CRISPR 101 ebook