Base Editing
Last update: Sep 2020
Base editing enables making single-base changes (point mutations) in cellular DNA or RNA (using Cas13b) without making DSBs, which reduces the number of editing by-products such as indels.
The technique often uses Cas9 D10A nickases fused with base deaminase enzymes. The Cas9 nickase component of the base‐editor protein targets the deaminase activity to the region of interest and makes a nick, while the deaminase allows base editing itself. Deaminase domains consist either of adenosine deaminase or cytosine deaminase, depending on the desired type of edit.
Deaminase domain acts on the target base, shown as the purple part of the non-target strand in Fig.1. If we use adenosine deaminase, adenosine base on the non-target strand will be converted to inosine (Fig.2). Simultaneously, the Cas9 nickase component makes a single strand break on the target strand, which activates the cellular repair mechanism. DNA repair pathway reads I as a G, and so it changes the T from the TI pair to CI. The pair is then resolved to the GC pair. In this way, base editing allowed AT to GC conversion at a specific site. GC to AT conversion happens similarly. In this pathway, cytosine deaminase converts cytosine to uracil.
The significance of developing base editing techniques is paramount. Since a large percentage of human pathogenic mutations is the results of single nucleotide mutations (single nucleotide polymorphisms), the possibility of exchanging a single base means that we could potentially treat many genetic disorders.
Figure 1. Base editing mechanism
While DSBs created by traditional CRISPR-Cas systems often result in indels, translocations, and rearrangements, base editing is a more precise tool, creating point mutations with high reliability and efficiency. Impressively, in human embryonic kidney cells and bone-cancer cells, base editing managed to make the desired edits with 50% efficiency and almost no detectable by-products. On the contrary, using a CRISPR-Cas based method employing a DNA repair template containing the desired base change made the corrections with less than 5% efficiency. On top of that, the traditional method often caused large indels.
While Cas9 nickases are usually used for base editing, Cas12a was also shown to produce a point mutation. In 2018, cytidine deaminase base edit was achieved in human cells using the dLbCpf1-BE0 base editor, which is a fusion of a rat APOBEC1 domain and uracil DNA glycosylase inhibitor to dLbCpf1. This base editor is effective in T-rich regions where Cas9 does not bind because of the differences in the PAM sequence.
Figure 2. Introducing point mutations by switching base pairs from TA to CG
Sources
Figure 1. G. Brett Robb. 2019. Genome Editing with CRISPR‐Cas: An Overview. Current Protocols Vol. 19, Issue 1, https://doi.org/10.1002/cpet.36
Figure 2. Gaudelli, N., Komor, A., Rees, H. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017). https://doi.org/10.1038/nature24644