Base Editing: Efficient Installation of Point Mutations with Minimal Byproducts

The majority of all mutations leading to human disease are attributed to point mutations, or single nucleotide polymorphisms (SNPs). Using base editing, an efficient genome editing strategy developed in our laboratory, we often observe ≥60% correction of SNPs in tissue culture without cell sorting or selection, along with minimal (0%–3%) undesired insertion or deletion byproducts [1]. Base editing is mediated by a cytidine [2] or evolved deoxyadenosine [3] deaminase that chemically modifies a target C or A nucleotide. When read by polymerases during transcription or DNA synthesis, the modified nucleotide is interpreted as another base, leading to the desired edit. Base-modifying enzymes are fused to catalytically impaired Cas9 to allow facile targeting to a desired genomic site without introducing double-stranded DNA breaks. The binding of a guide RNA to a target DNA locus creates a small bubble of single-stranded genomic DNA displaced by the guide RNA. Base editors perform their chemical surgery on individual bases within this single-stranded DNA bubble.

Since its development 2 years ago, base editing has been successfully used in a variety of cell types and animal models, including insects, frogs, fish, mice, and human embryos. Delivery has been achieved using viruses [4,5], lipid nanoparticles [6], hydrodynamic injection [7], and embryo injection [8 –10] of editor plasmid DNA, messenger RNA, and protein. The advantages of using base editors over using Cas9-mediated homology-directed repair (HDR) to install desired edits are (1) no exogenous template need be supplied, (2) efficiency is generally much higher, especially in nondividing cells, and (3) undesired byproducts are much rarer because base editing does not normally create double-strand DNA breaks that trigger indels [11], translocations [12], large deletions [11], rearrangements [13], or p53 activation [14]. Disadvantages of base editors relative to Cas9-mediated HDR include (1) the larger size of base editors relative to Cas9, complicating its efficient packaging into some viral vectors, and (2) base editors are limited to the installation of point mutations (currently, the transition mutations, C ↔ T or A↔G), whereas HDR can in principle convert any sequence into virtually any other sequence. Nevertheless, because transition mutations are the most common types of pathogenic SNPs, current base editor tools can in principle install or correct a majority of known pathogenic human SNPs [3]. Future work to expand the base chemistry and targeting scope of base editors will further enhance their potential.

Efficient delivery in vivo remains a significant challenge for all potential genome editing therapeutics. Blood disease targets are among the most attractive systems for the development of base editor therapeutics because of established protocols for ex vivo modification and transplantation of hematopoietic cells into patients. In the near future, a variety of mutations associated with blood diseases may be corrected by base editing and evaluated in animal models as starting points for the development of new human therapeutics.

My mentor David Liu has fostered an outstanding training environment by focusing his team on the greatest needs that face medicine and biotechnology. He supports bold, creative, and ambitious projects with thoughtful direction. He serves as a hub for countless collaborations, connecting experts that together can accomplish much more than any could on their own. David's tireless work inspires his laboratory to drive the development and application of much-needed tools until they have reached their fullest potential. When I transition to the next stage of my career and take on a mentorship role, I will strive to continue this devotion to multidisciplinary work and support my trainees' endeavors to apply creative solutions to the world's most pressing problems.