CRISPR 101: Cytosine and Adenine Base Editors

Early CRISPR applications were often limited by the low editing efficiency of homology-directed repair (HDR), the pathway for resolving DNA double-strand breaks (DSBs) preferred by researchers. Compared to non-homologous end joining (NHEJ), HDR occurs at a relatively low frequency, especially in nondividing cells. Scientists everywhere wistfully imagined being able to specify and correct single-base mutations without introducing DSBs at all.

Base editing to the rescue!

Base editors chemically modify DNA bases without breaking the DNA backbone, sidestepping the problem of HDR vs. NHEJ entirely. The first two classes of base editors were cytosine base editors (CBEs) and adenine base editors (ABEs). CBEs mediate a C to T change (or a G to A change on the opposite strand). ABEs make an A to G change (or a T to C change on the opposite strand). Both are base transition editors: converting one purine base to the other or one pyrimidine base to the other.

Figure 1: Base transition editors catalyze purine-to-purine or pyrimidine-to-pyrimidine base changes. Solid red arrows indicate a conversion directly catalyzed by the base editor. Dotted red arrows indicate the change introduced to the opposite strand following base editing and DNA repair. Black arrows indicate natural cellular processes. BER = base excision repair. Created with BioRender.com.

How does base editing work?

Base transition editing requires three elements. They are, broadly:

1. A Cas protein (catalytically dead or nickase) fused to a deaminase that makes the edit
2. A gRNA targeting Cas to a specific locus
3. A target base for editing within the editing window specified by the Cas protein

These elements were the starting point towards the development of the first cytosine base editors from Alexis Komor and the first adenine base editors from Nicole Gaudelli in David Liu’s lab in 2016.

History of base transition editing

Cytosine base editing

The beginnings

Komor created the first cytosine base editor by coupling a cytidine deaminase with the inactive dCas9 (Komor et al., 2016). These fusions convert cytosine to uracil without cutting DNA. Uracil is then subsequently converted to thymine through DNA replication or repair. Fusing an inhibitor of uracil DNA glycosylase (UGI) to dCas9 prevents base excision repair, which would change the U back to a C mutation.

To increase base editing efficiency, Komor and team wanted to force the cell to use the deaminated DNA strand as a template. To do so, they used a Cas nickase, instead of dCas9. The resulting editor, BE3, nicks the unmodified DNA strand so that it appears “newly synthesized” to the cell. Thus, the cell repairs the DNA using the U-containing strand as a template, copying the base edit.

Figure 2: Cytidine deamination takes place on the free strand of DNA and converts a C to U without producing a double strand break. Created with BioRender.com.

The BE3 system increased editing frequency to above 30% for a variety of targets in human cell lines, with an average indel frequency of only 1.1%. These numbers are a vast improvement over Cas9-mediated HDR for the loci tested, where average HDR-mediated editing frequency was only 0.5%, and more indels were observed than point modifications. CRISPR base editing persists through multiple cell divisions, indicating that this method produces stable edits. However, this system is also subject to off-target editing based on Cas9 off-target activity. 

Find CRISPR base editing plasmids at Addgene

Improving cytosine base editing scope and efficiency

Since the development of BE3, many research groups have made improvements to base editors, including:

• expanding targeting scope
• improving editing efficiency
• decreasing off-target effects

In 2016, Akihiko Kondo’s lab created the Target-AID base editor using a cytidine deaminase from sea lamprey fused to Cas9 nickase (Nishida et al., 2016). Target-AID acts similarly but not identically to BE3, modifying a 3-5 base window 18 bases upstream of the PAM. 

David Liu’s lab generated BE3 variants with other deaminases: AID, CDA1, and APOBEC3G (Komor et al., 2017). CDA1-BE3 and AID-BE3 edited Cs following a G more efficiently than BE3, but APOBEC3G displayed less predictable sequence preferences. 

The Liu lab also used natural and engineered Cas9 variants to develop five new base editors with distinct PAM sequences, expanding the number of available target sites for base editing (Kim et al., 2017). For each base editor, they observed editing activity with a minimum efficiency of ~50% and confirmed that the fusion protein retained the PAM properties of the individual Cas9. They also mutagenized the cytidine deaminase portion of the base editor to create SpCas9 base editors with editing windows as small as 1-2 nucleotides.

To reduce off-target effects associated with base editing, the lab created HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3 showed 37-fold less off-target editing than BE3, with only a slight reduction in on-target editing efficiency. To further improve specificity, they purified HF-BE3 protein for delivery in ribonucleoprotein particles (RNPs) to both zebrafish embryos and the mouse inner ear.

Fourth-generation cytosine base editors

The fourth-generation base editors, BE4, reduce the undesired C→G or C→A conversions that can happen with BE3s. These byproducts likely resulted from excision by uracil N-glycosylase (UNG) during base excision repair. Adding a second copy of the UNG inhibitor, UGI, increases base editing product purity. The lab also extended the APOBEC1-Cas9n and Cas9n-UGI linkers to improve product purity, and these three improvements represent the fourth generation of base editors. Compared to BE3, BE4 offers a 2.3 fold decrease in C→G and C→A products as well as a 2.3 fold decrease in indel formation.

To further decrease indel formation 1.5-2 fold, the team fused bacteriophage protein Gam to the N-terminus of BE4 (Komor et al., 2017). Gam binds the free ends of DSBs, which may lead to cell death rather than NHEJ repair, thus removing these cells from the edited population. 

Another way to improve base editing efficiency for mammalian edits is to ensure the editors make it into the nucleus and that they are expressed well. The Liu lab modified the nuclear localization signals and codon usage BE4 to create BE4max and AncBE4max with a 4.2-6-fold improvement in editing efficiency (Koblan et al., 2018). Later directed evolution efforts yielded an editor with more flexible sequence preferences, evoAPOBEC1-BE4max, which is significantly more effective at editing cytosines preceded by guanine (Thuronyi et al., 2019).

Following the development of adenine base editors (discussed next!), RNA adenine deaminases have been engineered and/or evolved to catalyze cytidine deamination instead of (or in addition to) adenosine deamination (Chen et al., 2023; Lam et al., 2023; Neugebauer et al., 2023). These CBEs (called Td-CBEs, CBE-Ts, or TadCBEs) are smaller and less likely to cause off-target mutations than those derived from naturally occurring cytidine deaminases.

Adenine base editors

The beginnings

Nicole Gaudelli from David Liu’s lab created an adenine base editor that would convert adenine to inosine, resulting in an A to G change (Gaudelli et al., 2017). Creating an adenine base editor required an additional step because there are no known DNA adenine deaminases. The team used directed evolution to create one from the RNA adenine deaminase TadA.  

After seven rounds of molecular evolution, they obtained four adenine base editors (ABEs). ABE7.10 is the most active editor, displaying an average editing efficiency of 53% with an editing window of target positions 4-7. ABEs 6.3, 7.8, and 7.9 display slightly wider editing windows of position 4-9, although editing efficiency may be lower at positions 8 and 9. While cytosine base editors often produce a mixed population of edits, ABEs do not display significant A to non-G conversion at target loci. The removal of inosine from DNA is likely infrequent, thus preventing the induction of base excision repair.

In terms of off-target effects, ABEs also compare favorably to other methods. In a head-to-head comparison with Cas9, Cas9 modified 9/12 known off-targets with a 14% indel rate, while ABE7.10 modified only 4/12 off-targets with a frequency of 1.2%. Although the lab did not conduct comprehensive genome-wide studies of ABE specificity, their other experiments suggest that ABEs are robust and specific editors.

Improving adenine base editing

When the Liu lab created BE4max and AncBE4max mentioned above, they also made an adenine base editor with improved nuclear localization and expression. This base editor was named ABEmax.

In 2020, two papers were published describing additional ABEs evolved from ABE7.10 that have improved base editor targeting flexibility and specificity. In this first, the Liu lab used phage-assisted evolution selection systems to generate ABE8e(TadA-8e V106W), which edits ~590-fold faster than the TadA from ABE7.10 without increasing off-target activity (Richter et al., 2020). This is important because ABEs have generally been slow, meaning that the Cas9 domain often lets go of the DNA before the edit is made.

Using ABE7.10 as a starting point, Gaudelli evolved the base editor into 40 new ABE8 variants (Gaudelli et al., 2020). Compared to ABE7.10, ABE8s resulted in 1.5-fold more editing at protospacer positions A5-A7 and 3.2-fold more editing at positions A3-A4 and A8-A10 at NGG PAM and 4.2-fold higher editing efficiency at non-NGG PAM variants compared to ABE7.10. ABE8s have an improved base editing capacity, even at sites previously difficult to target. ABE8s can achieve 98-99% target modification in primary T cells, making them a promising tool for cell therapy applications.

Dual base editors

What about combining the function of both editors into one?

This has been done by fusing the adenine and cytosine editing components together. Keith Joung’s lab created a dual-deaminase editor called SPACE (synchronous programmable adenine and cytosine editor) by fusing miniABEmax-V82G and Target-AID to Cas9 (Grünewald et al., 2020). Another lab took a similar approach. Dali Li’s lab’s A&C-BEmax consists of a fusion of both cytidine and adenosine deaminases with a Cas9 nickase (Zhang et al., 2020). It has increased CBE activity and reduced RNA off-targeting activity compared to ABEmax.

In an alternative approach, engineering and directed evolution of the TadA domain has yielded base editors that are equally efficient at cytosine and adenine base editing, called CABE-Ts and TadDEs without additional fused components (Lam et al., 2023; Neugebauer et al., 2023).

What other editing strategies are there?

CBEs and ABEs can induce 4 out of 12 possible base-to-base changes. The other 8 base-to-base changes require Base Transition Editors, which will be discussed in another post.

Base editors have a narrow editing window of only a few nucleotides, so they are tightly constrained by PAM availability in the genome. So the development of near-PAMless Cas9 variants significantly expands the library of accessible genomic targets for base editors (Walton et al., 2020).

There are also RNA base editors, which you can learn more about in this blog post: CRISPR 101: RNA Editing with Cas13.

Finally, the development of prime editing, which uses a different mechanism than base editing, has allowed scientists to replace entire sequences of varying lengths.

Base editing publication highlights

In the table below, you'll find a list of publications we've highlighted in this article. 

Publication	Plasmids		Highlights
Komor et al., 2016	BE1, BE2, BE3		BE3 displays highest editing efficiency but higher indel formation than BE2
Nishida et al., 2016	Target-AID		Edits 3-5 base window surrounding -18 position upstream of the PAM
Kim et al., 2017	SaBE3, BE3 PAM variants, BE3 editing window variants		Greatly expands the number of target loci for base editing
Rees et al., 2017	HF-BE3		HF-BE3 and ribonucleoprotein delivery decrease BE3 off-target activity
Komor et al., 2017	BE4 and BE4-Gam; AID, CDA1 and APOBEC3G BE3 variants		A second copy of UGI improves product purity. Gam decreases indel frequency.
Koblan et al., 2018	BE4max, ArcBe4max, ABEmax		Improved nuclear localization and expression
Gaudelli et al., 2017	Adenine base editors (ABE7.10)		A→I (A→G) editing with high product purity and low off-target editing
Thuronyi et al., 2019	evoAPOBEC1-BE4max		Improved editing of Cs preceded by Gs
Richter et al., 2020	ABE8		590x faster adenine base editing than ABE7.10
Gaudelli et al., 2020	ABE8e		Efficient editing in primary cells
Zhang et al., 2020	A&C-BEmax		Fused cytosine and adenine base editors
Grünewald et al., 2020	SPACE		Fused cytosine and adenine base editors
Walton et al., 2020	SpRY Cas9, available fused to ABEmax(7.10) or to BE4max		Evolved SpCas9 with NRN or NYN PAM site
Chen et al., 2023	Td-CBE		Engineered TadA domain for cytosine base editing
Lam et al., 2023	CBE-T, CABE-T		Engineered TadA domain for cytosine base editing (CBE-T) or dual cytosine and adenine base editing (CABE-T)
Neugebauer et al., 2023	TadCBE, TadDE		Evolved TadA domain for cytosine base editing (TadCBE) or dual cytosine and adenine base editing (TadDE)

This post was originally written by Mary Gearing in August 2016 and was updated by Jennifer Tsang in August 2020 and by Emily P. Bentley in February 2025.

---

References and Resources

References

Chen, L., Zhu, B., Ru, G., Meng, H., Yan, Y., Hong, M., Zhang, D., Luan, C., Zhang, S., Wu, H., Gao, H., Bai, S., Li, C., Ding, R., Xue, N., Lei, Z., Chen, Y., Guan, Y., Siwko, S., … Li, D. (2023). Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. Nature Biotechnology, 41(5), 663–672. https://doi.org/10.1038/s41587-022-01532-7

Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., & Liu, D. R. (2017). Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature, 551(7681), 464–471. https://doi.org/10.1038/nature24644

Gaudelli, N. M., Lam, D. K., Rees, H. A., Solá-Esteves, N. M., Barrera, L. A., Born, D. A., Edwards, A., Gehrke, J. M., Lee, S.-J., Liquori, A. J., Murray, R., Packer, M. S., Rinaldi, C., Slaymaker, I. M., Yen, J., Young, L. E., & Ciaramella, G. (2020). Directed evolution of adenine base editors with increased activity and therapeutic application. Nature Biotechnology, 38(7), 892–900. https://doi.org/10.1038/s41587-020-0491-6

Grünewald, J., Zhou, R., Lareau, C. A., Garcia, S. P., Iyer, S., Miller, B. R., Langner, L. M., Hsu, J. Y., Aryee, M. J., & Joung, J. K. (2020). A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing. Nature Biotechnology, 38(7), 861–864. https://doi.org/10.1038/s41587-020-0535-y

Kim, Y. B., Komor, A. C., Levy, J. M., Packer, M. S., Zhao, K. T., & Liu, D. R. (2017). Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nature Biotechnology, 35(4), 371–376. https://doi.org/10.1038/nbt.3803

Koblan, L. W., Doman, J. L., Wilson, C., Levy, J. M., Tay, T., Newby, G. A., Maianti, J. P., Raguram, A., & Liu, D. R. (2018). Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nature Biotechnology, 36(9), 843–846. https://doi.org/10.1038/nbt.4172

Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533(7603), 420–424. https://doi.org/10.1038/nature17946

Komor, A. C., Zhao, K. T., Packer, M. S., Gaudelli, N. M., Waterbury, A. L., Koblan, L. W., Kim, Y. B., Badran, A. H., & Liu, D. R. (2017). Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Science Advances, 3(8), eaao4774. https://doi.org/10.1126/sciadv.aao4774

Lam, D. K., Feliciano, P. R., Arif, A., Bohnuud, T., Fernandez, T. P., Gehrke, J. M., Grayson, P., Lee, K. D., Ortega, M. A., Sawyer, C., Schwaegerle, N. D., Peraro, L., Young, L., Lee, S.-J., Ciaramella, G., & Gaudelli, N. M. (2023). Improved cytosine base editors generated from TadA variants. Nature Biotechnology, 41(5), 686–697. https://doi.org/10.1038/s41587-022-01611-9

Neugebauer, M. E., Hsu, A., Arbab, M., Krasnow, N. A., McElroy, A. N., Pandey, S., Doman, J. L., Huang, T. P., Raguram, A., Banskota, S., Newby, G. A., Tolar, J., Osborn, M. J., & Liu, D. R. (2023). Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nature Biotechnology, 41(5), 673–685. https://doi.org/10.1038/s41587-022-01533-6

Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., Mochizuki, M., Miyabe, A., Araki, M., Hara, K. Y., Shimatani, Z., & Kondo, A. (2016). Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science (New York, N.Y.), 353(6305), aaf8729. https://doi.org/10.1126/science.aaf8729

Rees, H. A., Komor, A. C., Yeh, W.-H., Caetano-Lopes, J., Warman, M., Edge, A. S. B., & Liu, D. R. (2017). Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nature Communications, 8(1), 15790. https://doi.org/10.1038/ncomms15790

Richter, M. F., Zhao, K. T., Eton, E., Lapinaite, A., Newby, G. A., Thuronyi, B. W., Wilson, C., Koblan, L. W., Zeng, J., Bauer, D. E., Doudna, J. A., & Liu, D. R. (2020). Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nature Biotechnology, 38(7), 883–891. https://doi.org/10.1038/s41587-020-0453-z

Thuronyi, B. W., Koblan, L. W., Levy, J. M., Yeh, W.-H., Zheng, C., Newby, G. A., Wilson, C., Bhaumik, M., Shubina-Oleinik, O., Holt, J. R., & Liu, D. R. (2019). Continuous evolution of base editors with expanded target compatibility and improved activity. Nature Biotechnology, 37(9), 1070–1079. https://doi.org/10.1038/s41587-019-0193-0

Walton, R. T., Christie, K. A., Whittaker, M. N., & Kleinstiver, B. P. (2020). Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science (New York, N.Y.), 368(6488), 290–296. https://doi.org/10.1126/science.aba8853

Zhang, X., Zhu, B., Chen, L., Xie, L., Yu, W., Wang, Y., Li, L., Yin, S., Yang, L., Hu, H., Han, H., Li, Y., Wang, L., Chen, G., Ma, X., Geng, H., Huang, W., Pang, X., Yang, Z., … Li, D. (2020). Dual base editor catalyzes both cytosine and adenine base conversions in human cells. Nature Biotechnology, 38(7), 856–860. https://doi.org/10.1038/s41587-020-0527-y

Additional Resources on the Addgene Blog

• CRISPR 101: Homology-Directed Repair
• Learn about base editing for plants
• Read about four base editor reporters

Additional Resources on Addgene.org

• Catch up on your CRISPR background with our CRISPR Guide
• Browse CRISPR plasmids for base editing
• Find validated gRNAs