Rees, H. A. | Search bioRxiv

bioRxiv2020

Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

The foundational adenine base editors (e.g. ABE7.10) enable programmable C*G to T*A point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG PAM sites, ABE8s result in [~]1.5x higher editing at protospacer positions A5-A7 and [~]3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a [~]4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the {gamma}-globin genes HBG1 and HBG2, with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification which is maintained when multiplexed across three loci. Delivered as mRNA, ABE8s induce no significant levels of sgRNA-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

genomics↗

bioRxiv2020

Next-generation cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity

Abstract/introductory paragraphCytosine base editors (CBEs) are molecular machines which enable efficient and programmable reversion of T*A to C*G point mutations in the human genome without induction of DNA double strand breaks1, 2. Recently, the foundational cytosine base editor (CBE) BE3, containing rAPOBEC1, was reported to induce unguided, genomic DNA3, 4 and cellular RNA5 cytosine deamination when expressed in living cells. To mitigate spurious off-target events, we developed a sensitive, high-throughput cellular assay to select next-generation CBEs that display reduced spurious deamination profiles relative to rAPOBEC1-based CBEs, whilst maintaining equivalent or superior on-target editing frequencies. We screened 153 CBEs containing cytidine deaminase enzymes with diverse sequences and identified four novel CBEs with the most promising on/off target ratios. These spurious-deamination-minimized CBEs (BE4 with either RrA3F, AmAPOBEC1, SsAPOBEC3B, or PpAPOBEC1) were further optimized for superior on- and off-target DNA editing profiles through structure-guided mutagenesis of the deaminase domain. These next-generation CBEs display comparable overall DNA on-target editing frequencies, whilst eliciting a 10- to 49-fold reduction in C-to-U edits in the transcriptome of treated cells, and up to a 33-fold overall reduction in unguided off-target DNA deamination relative to BE4 containing rAPOBEC1. Taken together, these next-generation CBEs represent a new collection of base editing tools for applications in which minimization of spurious deamination is desirable and high on-target activity is required.

genomics↗