Journal: Nature communications

Article Title: Engineering and optimising deaminase fusions for genome editing.

doi: 10.1038/ncomms13330

Figure Lengend Snippet: 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. *Pvalueo0.05, **Pvalueo0.01, ***Pvalueo0.001, n ¼ 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. *Pvalueo0.05, **Pvalueo0.01, ***Pvalueo0.001, n ¼ 4). (e) GFP rescue by ZF-AIDs and TALE-AID in (wild type), (Dung), and (DmutS Dung) strains. All error bars indicate s.d. (All t-tests compare the fusion deaminases against the AID control. *Pvalueo0.05, **Pvalueo0.01, ***Pvalueo0.001, n ¼ 4). (f) E.coli (DmutS Dung) cells imaged under fluorescence (upper) and phase contrast (lower) after expression of ZF-AID or AID for 10 h. Top, Scale bar, 20 mm. 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.

Article Snippet: GFP-In reporter cells were plated in 12-well plates the day before transfection at densities of 4 105 cells per well transfected using Lipofectamine 2000 (Invitrogen) and 1.6 mg DNA (400 ng of ZF-AID/ZF-nuclease/ I-Sce1 expression plasmid and/or 20 ng of UGI expression plasmid, and/or 20 ng of ShRNA-MSH6 expression plasmid, 1 mg of DNA donor pUC19 (Invitrogen) plasmid to 1.6 ug) per well.

Techniques: Activity Assay, Binding Assay, Sequencing, Transformation Assay, Plasmid Preparation, Expressing, Cytometry, Control

Journal: Nature communications

Article Title: Engineering and optimising deaminase fusions for genome editing.

doi: 10.1038/ncomms13330

Figure Lengend Snippet: 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-AIDDNES 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 DNES/UGI/sr1 or ZF GFPINL-AIDDNES/UGI/sr1 plasmids. Scale bar, 200 mm. (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, ZFGFPINNs and ZFGFPIN-AIDDNESs binding sites (ZF GFPINNs/ZFGFPIN-AIDDNESs_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/ZFGFPINNs/ZFGFPIN-AIDDNESs 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 ZFGFPIN-AID/UGI relative to I-Sce1. A value of o1 shows decreased cell survival as compared with I-SceI, and demonstrates a toxic effect.

Article Snippet: GFP-In reporter cells were plated in 12-well plates the day before transfection at densities of 4 105 cells per well transfected using Lipofectamine 2000 (Invitrogen) and 1.6 mg DNA (400 ng of ZF-AID/ZF-nuclease/ I-Sce1 expression plasmid and/or 20 ng of UGI expression plasmid, and/or 20 ng of ShRNA-MSH6 expression plasmid, 1 mg of DNA donor pUC19 (Invitrogen) plasmid to 1.6 ug) per well.

Techniques: Activity Assay, Transfection, Construct, shRNA, Binding Assay, Sequencing, Expressing, Cytometry, Negative Control, Cytotoxicity Assay