An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities

Abstract

Base editor technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine DNA alterations. However, existing base editors create unwanted C-to-T alterations when more than one C is present in the enzyme's five-base-pair editing window. Here we describe a strategy for reducing bystander mutations using an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN hierarchy. In direct comparisons with the widely used base editor 3 (BE3) fusion in human cells, our eA3A-BE3 fusion exhibits similar activities on cytidines in TC motifs but greatly reduced editing on cytidines in other sequence contexts. eA3A-BE3 corrects a human β-thalassemia promoter mutation with much higher (>40-fold) precision than BE3. We also demonstrate that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites.

Figure 1:. Engineering and characterization of an A3A-BE3 base editor that selectively edits Cs preceded by a 5’ T.

(a) Schematic illustrating the architecture of the original BE3 fusion (consisting of rAPO1 linked to SpCas9 nickase and UGI) and the A3A-BE3 fusion. (b) Activities of BE3, A3A-BE3, and a series of A3A-BE3 variants bearing mutations in A3A on an integrated EGFP reporter gene target site bearing a cognate cytidine preceded by a 5’ T and a bystander cytidine preceded by a 5’ G in the editing window. Center values represent the mean of n = 3 biologically independent samples and error bars represent SEM .(c) Schematic summarizing specific and non-specific interactions between amino acid positions in A3A and its substrate single-stranded DNA derived from previously published co-crystal structures. (d) Heat maps showing C-to-T editing efficiencies for BE3, YE BE3s, and various A3A-BE3 variants at 12 endogenous human gene target sites, each bearing a cognate cytidine preceded by a 5’ T (indicated with a black arrow) and one or more bystander cytidines within the editing window. All editing efficiencies shown represent the mean of n = 3 biologically independent samples.

Figure 2:. Off-target editing activities of BE3 and eA3A-BE3 variants.

On- and off-target editing frequencies of four gRNAs targeted to (a) EMX1 site 1, (b) VEGFA site 2, (c) FANCF site 1 or (d) CTNNB1 site 1 with BE3 or one of the indicated eA3A-BE3 variants. Percentage edits represent the sum of all edited Cs in the editing window and represent the mean of n = 3 biologically independent samples with error bars representing SEMs. Intended target sequence is shown at the top of each graph. On-target sites are marked with a black diamond to the left and mismatches or bulges in the various off-target sites are shown with colored boxes or a dash in gray boxes, respectively. Off-target sites that lose the cognate TC motif within the editing window and thus might be expected to show lower off-target editing by eA3A, are noted with empty circles to the left. Asterisks indicate statistically significant differences in editing efficiencies observed between BE3 and eA3A-BE3 at each site (* p < 0.05, ** p < 0.005, *** p < 0.0005). All statistical testing was performed using two-tailed Student’s t-test according to the method of Benjamini, Krieger, and Yekutieli without assuming equal variances between samples.

Figure 3:. On- and off-target activities of eA3A-BE3 variants at a β-thalassemia-causing mutation HBB −28 (A>G) sequence in human cells.

(a) Schematic of the HBB −28 (A>G) mutation and potential base editing outcomes when targeting Cs at −28 and −25 in the editing window of an HBB-targeting gRNA. Mutations to the bystander cytidine at the −25 position are deleterious and cause β-thalassemia phenotypes independent of the identity of the −28 nucleotide. (b) Heat maps showing C-to-T editing efficiencies for BE3, YE BE3s, and various A3A-BE3 variants at the HBB −28 (A>G) target site in an integrated reporter in human HEK293T cells. The −28 C is indicated with a black arrow. Editing efficiencies shown represent the mean of n = 3 biologically independent samples. (c) Graph showing the frequencies of perfectly corrected (−28 C to T only) and other imperfectly edited (−28 C to G or other edited Cs) alleles by BE3, YE BE3 variants, and eA3A-BE3 variants. Efficiencies shown represent the mean of n = 3 biologically independent samples. (d) On- and off-target editing frequencies of the HBB-targeted gRNA with BE3, YE BE3 variants, or eA3A-BE3 variants. Percentage edits represent the sum of all edited Cs in the editing window and represent the mean of n = 3 biologically independent samples with error bars representing SEMs. Intended target sequence is shown at the top. On-target site is marked with a black diamond to the left and mismatches or bulges in the various off-target sites are shown with colored boxes or a dash in gray boxes, respectively. Off-target sites that lose the cognate TC motif within the editing window and thus might be expected to show lower off-target editing by eA3A, are noted with empty circles to the left. Asterisks indicate statistically significant differences in editing efficiencies observed between BE3 and eA3A-BE3 and between eA3A-BE3 and the untransfected control (* p < 0.05, ** p < 0.005, *** p < 0.0005). All statistical testing was performed using two-tailed Student’s t-test according to the method of Benjamini, Krieger, and Yekutieli without assuming equal variances between samples.