ARTICLE Received 13 May 2016 | Accepted 23 Sep 2016 | Published 2 Nov 2016 | Updated 9 Oct 2017 DOI: 10.1038/ncomms13330 OPEN Engineering and optimising deaminase fusions for genome editing Luhan Yang1,2,3,w, Adrian W. Briggs1,*, Wei Leong Chew1,2,*,w, Prashant Mali1, Marc Guell1,3,w, John Aach1, Daniel Bryan Goodman1,4, David Cox4, Yinan Kan1,3,w, Emal Lesha1,3,w, Venkataramanan Soundararajan1, Feng Zhang5,6,7 & George Church1,4,8 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. 1 Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. 2 Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02115, USA. 3 eGenesis Inc., 1 Kendal Square, Building 200, Cambridge Biolabs, Cambridge, Massachusetts 02139, USA. 4 Harvard-MIT Division of Health Science and Technology, Cambridge, Massachusetts 02139, USA. 5 Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA. 6 McGovern Institute for Brain Research, MIT, Cambridge, Massachusetts 02139, USA. 7 Department of Brain and Cognitive Sciences, MIT Cambridge, Cambridge, Massachusetts 02139, USA. 8 Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts 02115, USA. w Present addresses: eGenesis Inc., 1 Kendall Square, Building 200, Cambridge Biolabs, Cambridge, Massachusetts 02139, USA (L.Y., M.G., Y.K. and E.L.); Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore 138672, Singapore (W.L.C). * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to L.Y. (email: [email protected]) or to G.C. (email: [email protected]). NATURE COMMUNICATIONS | 7:13330 | DOI: 10.1038/ncomms13330 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms13330 enetic modification of mammalian cells has been greatly GFP-positive cells quantifiable by flow cytometry. Among the facilitated by the development of customized zinc finger four deaminase domains we tested, AID induced the highest GFP G(ZF)1,2, transcription activator-like effectors (TALEs)3 correction at the ACG site (Fig. 1c), thus the ZF-AID fusion was nucleases4,5 and clustered regularly interspaced short used for subsequent characterizations. No signal was observed in palindromic repeat/Cas9 (CRISPR/Cas9 (refs 6–8). These the control whereby only the ZF domain was expressed, programmable nucleases create targeted genomic double-strand indicating that GFP expression is attributed to the deaminase breaks (DSBs) that enhance gene conversion from exogenous activity. We confirmed the intended ACG-to-ATG conversion in homology donors via homology-dependent repair (HDR). 20/20 randomly chosen GFP þ bacterial colonies from the However, HDR faces numerous limitations: First, the nuclease- ZF-AID fusion, as assessed by Sanger sequencing. Therefore, induced DSBs are associated with genomic aberrations and ZF-AID introduces C-to-T mutations at the locus specified by the cytotoxicity9, which is more concerning when targeting multiple fused DNA-binding module. We do not exclude the potential genomic loci10. Second, HDR is highly inefficient compared with activities of other deaminase domains as ACG is not in the the competing non-homologous end joining pathway11,12, which preferred deaminase sites for some of them. results in generation of random insertions/deletions (indels) We next asked if we could reprogram the targeting site of AID instead of the intended genetic modifications. Third, in vivo with different DNA binding module. To achieve this, we genome editing remains challenging due to the difficulty in engineered a TALE-AID fusion (recognizing a different binding delivering donor DNA at sufficient concentration and the low sequence 50-TCACGATTCTTCCC-30 (ref. 20) as reported in HDR activity in somatic cells13. previous studies) and a corresponding reporter E. coli strain Deaminases are naturally occurring proteins that operate (Fig. 2c). Induction of TALE-AID for 10 h led to GFP expression in various important cellular processes. Activation induced in 0.02% of the reporter population, lower than that from deaminase (AID) and apolipoprotein B mRNA editing enzyme ZF-AID, but nonetheless significantly higher than with TALE or 14 catalytic polypeptide-like family proteins (APOBECs) are AID expression alone (t-test, two-tailed, P(TALE-AID, TALE) ¼ 0.0069, cytidine deaminases critical to antibody diversification and P(TALE-AID, AID) ¼ 0.0186; n ¼ 4) (Fig. 1d). Importantly, GFP innate immunity against retroviruses14. These enzymes convert expression is dependent on correct sequence recognition, because cytidines (C) to uracils (U) in DNA. If DNA replication occurs TALE-AID and ZF-AID do not induce GFP expression in before uracil repair, the replication machinery will treat the uracil reporter E. coli cells lacking the cognate target sequences (Fig. 1d). as thymine (T), leading to a C:G to T:A base pair conversion15. Additional target DNA sequences do not increase editing This elegant editing mechanism suggests a simple and effective efficiency, suggesting that a single ZF-AID or TALE-AID is genome editing tool that might help us circumvent the limitations sufficient for editing (Supplementary Fig. 2 and Supplementary associated with nuclease-based approaches. Note 8). Thus, ZF-AID and TALE-AID converts C-to-T at Recently, programmable deamianses by fusing APOBECs with sequence-defined genomic loci. catalytically dead and nicking Cas9 have been reported16. This The results demonstrated feasibility of using programmed finding holds great promise for therapeutic editing due to its high deaminases for genome editing, but editing efficiency was low. on-target efficiency and low indel rate. However, its tendency to We reasoned that the endogenous uracil repair pathways could induce off-target deamination has not been characterized reverse the targeted deamination, which would limit the desired systematically. Here, we aim to test if we could modulate the C-to-T conversion. Therefore, we knocked out mutS and ung, two specific activities of chimeric deaminases and examine its genes critical for uracil repair. Editing by ZF-AID increased to specificity both near the target site and in the global level. 0.5% (five-fold) in the DmutS knockout, and to 3.5% (35-fold) in Here, we demonstrated that programmable deaminases the DmutS Dung double knockout (Fig. 1e). Similarly, editing by could be generated by fusing cytidine deaminases with the ZF TALE-AID increased to 0.1% (seven-fold increase) in the DmutS or TALE-DNA binding modules. They could site-specifically Dung knockout (Fig. 1e). We confirmed GFP fluorescence signal convert cytidines to thymidines with 13% efficiency in Escherichia by microscopy (Fig. 1f) and confirmed C-to-T transitions by coli and 2.5% in human cells under optimized conditions. sequencing the gfp gene of 20 randomly chosen GFP þ colonies However, off-target deaminations were detected both near the from both the ZF-AID- and TALE-AID-induced populations. target sites and genome-wide, indicating significant enzyme Hence, suppression of uracil repair, as well as the mismatch repair processivity and off-target activity. Future engineering is required pathways increases editing frequencies from the targeted to increase the specificity of programmable deaminases for deaminases. All subsequent experiments in E.coli were done in therapeutic applications. the DmutS Dung background. Optimization of targeted deaminases. We next conducted Results structural optimization of the targeted deaminases by varying Design of targeted deaminases. To test this, we first engineered linker lengths and sequence compositions21,22 (Fig. 2a). While targeted deaminases by fusing each candidate deaminase (human tested variants all led to robust GFP rescue, with ZF-8-aa-AID APOBEC1, APOBEC3F, APOBEC3G (2K3A) (ref. 17) and AID) achieving 7.5% GFP þ frequency after 10 h (Fig. 2b) and 13% with a sequence-specific ZF, which has proven to be effective after 30 h of induction (Supplementary Fig. 3 and Supplementary by previous studies (recognizing the 9bp DNA sequence Note 9). Sequence composition of the linker also influences 50-GCCGCAGTG-30 (ref. 18) (Fig. 1a). On the basis of available editing frequencies (t-test, two tailed, P ¼ 0.0032 and n ¼ 4). structures of APOBEC2, we tethered the ZF to the N-terminus of Hence, the linker determines performance of the overall the deaminases to prevent steric hindrance to catalysis, separated construct.