| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | Constructs used in this study were obtained by USER assembly, Gibson assembly, or synthesized by Genscript. Gene fragments used for PCR were purchased as mammalian codon-optimized gene fragments from IDT. PCR were performed with primers ordered from IDT using either Phusion U DNA Polymerase Green MultiPlex PCR Master Mix (ThermoFisher) or Q5 Hot Start High-Fidelity 2x Master Mix (New England Biolabs). Endo-free plasmids used for mammalian transfection were prepped using ZymoPURE II Plasmid Midiprep (Zymo Research Corporation) from 50 mL Mach1 (ThermoFisher) culture. Sequences for CBEs, protospacer sequences for sgRNA, and oligos used in this study can be found in Supplementary Table 1-4. | Get A Quote |
Cytosine 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 CB... More
