CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA

The all-protein cytosine base editor DdCBE uses TALE proteins and a double-stranded DNA-specific cytidine deaminase (DddA) to mediate targeted C•G-to-T•A editing. To improve editing efficiency and overcome the strict TC sequence-context constraint of DddA, we used phage-assisted non-continuous and continuous evolution to evolve DddA variants with improved activity and expanded targeting scope. Compared to canonical DdCBEs, base editors with evolved DddA6 improved mitochondrial DNA (mtDNA) editing efficiencies at TC by 3.3-fold on average. DdCBEs containing evolved DddA11 offered a broadened HC (H = A, C or T) sequence compatibility for both mitochondrial and nuclear base editing, increasing average editing efficiencies at AC and CC targets from less than 10% for canonical DdCBE to 15–30% and up to 50% in cell populations sorted to express both halves of DdCBE. We used these evolved DdCBEs to efficiently install disease-associated mtDNA mutations in human cells at non-TC target sites. DddA6 and DddA11 substantially increase the effectiveness and applicability of all-protein base editing.


Supplementary Information
Continuous evolution of mitochondria base editors with improved activity and expanded targeting scope Beverly Y. Mok 1,2,3 , Anna V. Kotrys 4,5 , Aditya Raguram 1,2,3 , Tony P. Huang 1,2,3 , Vamsi K. We previously performed a PANCE of canonical T7-DdCBE using a strains 9 and 10 transformed with MP6. These strains expressed the GCA or GCG linker, respectively (Extended Data Fig. 7a). After fifteen passages, the overnight fold phage propagation increased to 1,000, representing >1,000-fold improvement. Clonal sequencing of phage isolated at the end of PANCE, however, revealed a stochastic frameshift mutation within the open reading frame encoding either the left-or right-half of T7-DdCBE (data not shown, available upon request). We speculated that the premature stop codons helped improve phage fitness by reducing the translational burden on the phage, thus increasing phage propagation in a manner that was independent of the deamination activity of DddA. Given that DddA11 already exhibited a broadened targeting scope and non-zero GC activity (Fig. 6b), we hypothesized that DddA11 could be a promising evolutionary stepping-stone to serve as a starting target for evolving DddA variants towards higher GC activity.
We initiated PANCE of T7-DdCBE containing DddA11 in duplicates using the same MP6-transformed strains 9 and 10. One replicate in PANCE-GCA and one replicate PANCE-GCG evolved 'cheaters' in which gIII was recombined into the phage genome. The PANCE schedules shown in Extended Data Fig. 7d are for the other replicates that do not contain gIII within the SP genome. We isolated six to eight plaques from each replicate after round 9 and round 12 for clonal sequencing. The mutation N1378S was strongly enriched in PANCE-GCA and PANCE-GCG. One replicate of PANCE-GCA also showed strong consensus for the additional mutations A1341I and P1394S (Supplementary Table 7).
We selected two strongly enriched genotypes (7.9.1 and 7.12.1) and two moderately enriched genotypes (7.12.2 and 7.12.3) for validation of mtDNA base editing activity in human cells (Extended Data Fig. 8a Fig. 8d and 8e).

Structure alignment of DddA to APOBEC3G
Our previous work identified ssDNA-specific APOBEC3G cytidine deaminase, which has an intrinsic 5'-CC preference 45 , as the closest structural relative to DddA. We aligned the catalytic domain of human APOBEC3G complexed with its ssDNA 5'-CCA substrate 46 with DNA-free DddA. The PACE-derived DddA variants DddA8 and DddA11 expanded the putative TC sequence preference to include AC and CC (Fig. 3b). These variants contained mutations A1341V, N1342S, G1344R and G1344S that are positioned within a loop that aligns most closely to loop 3 of APOBEC3G (Extended Data Fig. 10a and 10b). Previous studies identified DNA-binding loop 3 to be critical for enhancing the catalytic activity of APOBEC3G at 5'CC 47 . In this study, the N1342S nucleotide substitution in DddA11e increased TC editing by 1.3-fold and yielded low but detectable AC and CC editing (Fig. 3f). These results suggest that the DddA loop containing N1342 could be engineered to improve the catalytic activity of DddA and support deamination at non-TC contexts.
In APOBEC3G, residue D317 in loop 7 is critical for selectivity towards C-1 47 (Extended Data Fig. 10b). Context-specific PANCE of DddA strongly enriched for E1370K across all tested linkers of ACC, CCC and GCC (Supplementary Table 3).
Given that loop 7 of APOBEC3G spatially aligns with the DddA loop containing E1370K, E1370K could also be involved in altering the substrate selectivity of DddA (Extended Data Fig. 10b).
The editing efficiencies for mismatched DdCBEs containing DddA variants were generally comparable to or resulted in 2-10% higher average editing than the equivalent non-mismatched DdCBE (see a and b). Given that DdCBEs containing DddA6 or DddA11 resulted in similar editing efficiencies when tested as a mismatched TALE or non-mismatched TALE, all subsequent figures, except for Figs. 2c, 2d, 3d and 3e, are produced from DdCBEs containing the original mismatched RVD. Values and error bars reflect the mean±s.d of n=3 independent biological replicates