David Liu’s lab created the first base editor in 2016 (Komor et al., 2016) and since then has been trying to expand their precision editing capabilities. Base editors make specific DNA base changes and consist of a catalytically impaired Cas protein (dCas or Cas nickase) fused to a DNA-modifying enzyme, in this case a deaminase. Base changes from C•G-to-T•A are mediated by cytosine base editors (CBEs) and base changes from A•T-to-G•C are mediated by adenine base editors (ABEs). How does this work? Through molecular biology teamwork. The guide RNA (gRNA) specifies the editing target site on the DNA, the Cas domain directs the modifying enzyme to the target site, and the deaminase induces the DNA base change without a DNA double-strand break. But base editors aren’t perfect. They may be slow, can only target certain sites, or make only a subset of base substitutions.
The need for speed: base editing before Cas lets go
|Figure 1: David Liu gives a talk at the Cell Symposia: Gene- and Cell-based Therapies: CRISPR, Stem Cells, and Beyond meeting earlier this month.|
Another issue that arises for base editors is that the editing domain must induce the base change before the Cas domain lets go of the DNA. ABEs are somewhat slow and need a strong Cas9 domain to “hold on” long enough to the DNA to induce a base change. Because of this, ABEs are only weakly compatible with many Cas domains beyond SpCas9. In order to expand the gene-editing applications and targeting scope of ABEs David Liu’s lab set out to evolve ABEs with an increased compatibility to Cas homologs.
Evolving better base editors with the help of phages!
The latest ABE from the Liu lab is ABE7.10 which contains the deoxyadenosine deaminase, TadA-7.10 (Gaudelli et al., 2017). The idea was to evolve a TadA-7.10 version with a faster deamination rate so that deamination can happen before Cas homologs let go. The Liu lab developed the Phage-Assisted Continuous Evolution (PACE) and Phage-Assisted Non-Continuous Evolution (PANCE) selection systems to engineer better and faster ABEs (Fig. 2). These systems allow for rapid continuous protein evolution through many rounds of mutations, selection, and replications per day.
The PACE and PANCE systems use M13 bacteriophage, which rely on the expression of gene III to create infectious particles. A plasmid-based genetic circuit in the bacteria links the expression of gene III to base editor activity and editing speed and consists of the following components:
- A plasmid in the host bacterium expressing dCas9
- A plasmid in the host bacterium that expresses gene III. Gene III is under the control of a T7 RNA polymerase and cannot be expressed until the base editor corrects early stop codons within T7 RNAP.
- A phage encoding the deaminase TadA-7.10
- A mutagenesis plasmid controlling the phage mutation rate
Phage infection results in a complete ABE protein. If the phage delivers an active variant of the base editor, a functional gene III product is produced and can infect bacterial cells. If a phage does not deliver an active variant, the phage is not infectious. The editing speed dictates which phage variants can propagate faster than the rate of dilution (passage from the infected to a fresh uninfected host cell culture). This dilution step represents the major difference between PACE and PANCE: for PANCE the phage progeny is not diluted out but gets manually passaged from an infected host-cell culture to an uninfected culture which results in less overall dilution and less stringent phage selection.
|Figure 2: Phage-assisted evolution of base-editing activity. A plasmid-based genetic circuit in the bacteria links the expression of gene III to base editor activity and editing speed.|
Introducing ABE8e - a fast and flexible adenine base editor
Lower-stringency PANCE, followed by higher stringency PACE, resulted in the evolution of TadA variants with much higher base editing activity compared to the original TadA-7.10 enzyme. After testing numerous TadA variants, they identified TadA-8e, which edits 590-fold faster than TadA-7.10. It is compatible with all Cas9 and Cas12 homologs that the researchers tested, including SpCas9, SaCas9, LbCas12a, enAsCas12a, SpCas9-NG, SaCas9-KKH, CP1028-SpCas9, and CP1041-SpCas9. The resulting base editor was named ABE8e. ABE8e is not only fast, it can also be multiplexed: when tasked to correct two point mutations that hamper the expression of the fetal hemoglobin gene, ABE8e made both changes simultaneously with 54% efficiency, about 7 times higher than ABE7.10.
However, the increased editing speed and targeting scope of ABE8e comes with a price: ABE8e showed increased off-target DNA and off-target RNA activity. To mitigate this drawback, the lab introduced an additional mutation (V106W) into TadA-8e and the resulting ABE8e(TadA-8e V106W) was as efficient in on-target DNA editing as ABE8e while maintaining the lower off-target DNA and off-target RNA editing rate of ABE7.10.
Why engineer and optimize base editors?
Targeted evolution of naturally occurring enzymes and functional domains speed up the development towards faster and more precise base editors capable of editing previously uneditable sequences. Four DNA base swaps, so called point mutations that change C to T, G to A, A to G, and T to C, represent about two-thirds of known pathogenic point mutations. Together, cytosine and adenine base editors can correct all four of these kinds of point mutations, in theory converting the disease-associated version of a gene back to the wild-type version. With the addition of ABE8e to the base editing toolbox, scientists can now make these edits much more efficiently and in more contexts.
Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464–471 . https://doi.org/10.1038/nature24644
Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR (2020) Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nature Biotechnology. https://doi.org/10.1038/s41587-020-0453-z
Additional resources on the Addgene blog
- Browse our blog posts on base editing
- Read more about CRISPR
- Learn all the basics about CRISPR in our CRISPR 101 blog series
Resources on Addgene.org