Engineering and optimising deaminase fusions for genome editing

Abstract

Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification is limited by the requirements for genomic lesions, homology donors and the endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases and test if we could introduce site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. Our programmable deaminases effectively convert specific cytidines to thymidines with 13% efficiency in Escherichia coli and 2.5% in human cells. However, off-target deaminations were detected more than 150 bp away from the target site. Moreover, whole genome sequencing revealed that edited bacterial cells did not harbour chromosomal abnormalities but demonstrated elevated global cytidine deamination at deaminase intrinsic binding sites. Therefore programmable deaminases represent a promising genome editing tool in prokaryotes and eukaryotes. Future engineering is required to overcome the processivity and the intrinsic DNA binding affinity of deaminases for safer therapeutic applications.

Figure 1. Design and targeted deaminase activity of chimeric deaminases in E.coli.

(a) Schematic representation of the design of targeted deaminases. The DNA binding domain (DBD), either ZF or TALE, was fused to the N-terminus of the deaminase with a certain linker. (b) Experimental overview: we integrated a GFP cassette (top) consisting of a broken start codon ACG, DNA binding sequence and the GFP coding sequence into the bacterial genome. We subsequently transformed targeted deaminases (middle) in pTrc-kan plasmid into the strain and induced protein expression. Targeted deamination of the C in the broken start codon leads to a ACG→ATG transition (bottom), rescuing GFP translation which is quantifiable via flow cytometry. (c) ZF-deaminases were tested for targeted deaminase activity by measuring GFP rescue. ZF, ZF-APOBECs (ZF-APOBEC1, ZF-APOBEC3F, ZF-APOBEC3G) or ZF-AID indicate cells transformed with plasmids that express ZF, ZF-APOBECs or ZF-AID respectively. All error bars indicate s.d. (All t-tests compare ZF-deaminases against the ZF control. *P_value<0.05, **P_value<0.01, ***P_value<0.001, n=4). (d) GFP rescue by ZF-AID and TALE-AID in the ZF-reporter and TALE-reporter strains.(All t-tests compare the fusion deaminases against the AID control. *P_value<0.05, **P_value<0.01, ***P_value<0.001, n=4). (e) GFP rescue by ZF-AIDs and TALE-AID in (wild type), (Δung), and (ΔmutS Δung) strains. All error bars indicate s.d. (All t-tests compare the fusion deaminases against the AID control. *P_value<0.05, **P_value<0.01, ***P_value<0.001, n=4). (f) E.coli (ΔmutS Δung) cells imaged under fluorescence (upper) and phase contrast (lower) after expression of ZF-AID or AID for 10 h. Top, Scale bar, 20 μm. More detailed structures and sequences of the fusion proteins and reporters are shown in Fig. 2a,c, Supplementary Fig. 1 and Supplementary Notes 1–7.

Figure 2. Optimization of targeted deamination frequency of AID fusions in E.coli.

(a) Schematic representation of ZF-AIDs variants tested for targeted deaminase activity (upper) and the reporter (lower) with the ZF-recognition sequence in blue. (b) GFP rescue by expression of the four ZF-AIDs variants and ZF or AID domains alone. All error bars indicate s.d. (c) Schematic representation of TALE-AIDs and the reporters tested for targeted deaminase activity. Five TALE-AIDs (upper) with different TALE C-terminus truncations (C1–C5) were constructed, with the remaining C-terminus lengths shown in parentheses. Full TALE-AID protein sequences can be found in Supplementary Note 2. Five reporters were constructed (lower) with different spacer lengths (2, 5, 8 and 11 bp) between the broken start codon and TALE DNA binding motif. The TALE binding site on the GFP reporter is shown in blue; the TALE N-terminus segment specifies the 5′ thymine base of the binding site. (d) All five TALE-AIDs were tested for targeted deaminase activity on all five reporters (c). Green and grey encode high and low GFP rescue, respectively.

Figure 3. Test of the specificity of AID fusions.

(a) Test of ZF-8aa-AID sequence specificity using a GFP reporter with point-mutated ZF binding sequences. t-tests compare each mutated site against the unmodified site (top). *P_value<0.05, **P_value<0.01, ***P_value<0.001, n=4. All error bars indicate s.d. (b) Test of TALE-C1-AID sequence specificity using a GFP reporter with point-mutated TALE binding sites. t-tests compare each mutated site against the unmodified site (top). *P_value<0.05, **P_value<0.01, ***P_value<0.001, n=4. All error bars indicate s.d. Note that we altered the first nucleotide, a TALE-N terminus-specified thymine, to three other nucleotides individually, while we changed other nucleotides in the TALE recognition domain to the nucleotide mostly likely to be recognized. (c) Mutation location and spectrum in the GFP gene of GFP+ and GFP− cells collected after ZF-8aa-AID induction. A schematic structure of the GFP gene is shown above the mutation frequency along the gene’s length among 200 Sanger sequenced colonies of each cell population. Grey lines indicate positions of C/G nucleotides; red lines indicate occurrences of the AID preferred motif (WRC). (d) Mutation spectrum on the GFP gene of GFP+ and GFP− cells collected after TALE-C1-AID induction. (e) Whole-genome SNV profiles of strains with/without ZF-AID induction. SNVs that may stem from cytosine deamination (C/G→T/A) are in either green (if C was in the AID-preferred WRC motif) or blue (all other Cs) bars. (f) Whole-genome SNVs profiles of strains with/without TALE-AID induction. Colour schematic is the same as e.

Figure 4. Targeted deamination and low toxicity of ZF-AID in human cells.

(a) Schematic representation of the ACG-GFP reporter system in HEK239FT cells (upper) and the ZF-AID (lower) tested for targeting deaminase activity. IRES, internal ribosome entry site; NLS, nuclear localization signal. (b) Targeted deamination activity of ZF-AIDs. ACG-GFP reporter cells were transfected with the constructs labelled on the X-axis. Targeted deamination frequency was estimated as the proportion of GFP-rescued cells 48 h after transfection. ZF-AID^ΔNES is identical to ZF-AID except with a deleted AID nuclear export signal (NES); UGI, inhibitor of UNG; sr1, shRNA-MSH2. (c) ACG-GFP reporter cells imaged under fluorescence (mCherry (left)/GFP (right)) 48 h after transfection with ZF-AID ^ΔNES/UGI/sr1 or ZF _GFPINL-AID^ΔNES/UGI/sr1 plasmids. Scale bar, 200 μm. (d) Schematic design of DSBs assay. The genomically integrated GFP-In reporter includes a 35 bp frame-shift insertion bearing a stop codon and I-SceI recognition site (I-SceI_RS). Of note, ZF_GFPINNs and ZF_GFPIN-AID^ΔNESs binding sites (ZF _GFPINNs/ZF_GFPIN-AID^ΔNESs_BS) were identical and located 82 bp upstream of the insertion. We transfected the cells with a DNA donor carrying the wild-type GFP sequence along with I-Sce1/ZF_GFPINNs/ZF_GFPIN-AID^ΔNESs expression plasmids and assessed the DSB-generating rate by measuring HDR frequency as determined by GFP rescue of the cells. (e) GFP rescue results determined by flow cytometry. Negative control was transfected with the DNA donor only. (f) Cytotoxicity assay for ZF_GFPIN-AID/UGI relative to I-Sce1. A value of <1 shows decreased cell survival as compared with I-SceI, and demonstrates a toxic effect.