Base Editor 3 (BE3), a cytosine base editor of rat APOBEC1, Cas9 D10A nickase, and uracil glycosylase inhibitor
Third-generation cytosine base editor that converts C·G base pairs to T·A within a window of about five nucleotides at the PAM-distal end of the protospacer, without creating double-strand breaks. It fuses rat APOBEC1 cytidine deaminase to Cas9 D10A nickase and uracil glycosylase inhibitor (UGI) through an XTEN linker.
Length: 5133 bp (1711 aa)
Type: Cytosine Base Editor
Conversion: C-to-T
Editing window: Positions 4-8
Origin: Rat APOBEC1 fused to Cas9 D10A nickase and UGI
Characteristics
The editor is rat APOBEC1, a 16-residue XTEN linker, Cas9 D10A nickase, and UGI. The deamination window spans about five nucleotides, typically positions 4 to 8 counting the PAM-distal end as position 1. UGI protects the edited uracil from excision, and nicking the non-edited strand raised editing 2- to 6-fold over BE2, which lacks the nickase, to up to 37% of total DNA sequences with the targeted C to T conversion in human cells. Indels averaged 1.1% across six loci, against 4.3% for wild-type Cas9 with a donor template, which converted C to T at only 0.5%. Editing is substantially lower when the base 5' of the target C is G.
Applications: Correction of disease-relevant point mutations by C to T conversion in transformed human and murine cell lines, including conversion of the APOE4 allele to APOE3r in astrocytes. Used to edit single bases without a donor template and without double-strand breaks.
Limitations: Converts every C within the deamination window, so neighboring cytosines are edited together; the editor processively converts most or all Cs in the window on the same strand. Needs an NGG PAM that places the target C at positions 4 to 8. Indels reached 4.6-6.1% at the APOE4 locus in astrocytes.
Architecture: Rat APOBEC1 cytidine deaminase, 16-residue XTEN linker, Cas9 D10A nickase, and uracil glycosylase inhibitor (UGI). The nickase cuts the non-edited strand to bias repair toward the edited base.
