Engineering and optimising deaminase fusions for genome editing.
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.
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.|Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02115, USA.|eGenesis Inc., 1 Kendal Square, Building 200, Cambridge Biolabs, Cambridge, Massachusetts 02139, USA.
Luhan
L
Yang
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Adrian W
AW
Briggs
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.|Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02115, USA.
Wei Leong
0000-0002-4774-7959
WL
Chew
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Prashant
P
Mali
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.|eGenesis Inc., 1 Kendal Square, Building 200, Cambridge Biolabs, Cambridge, Massachusetts 02139, USA.
Marc
M
Guell
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
John
J
Aach
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.|Harvard-MIT Division of Health Science and Technology, Cambridge, Massachusetts 02139, USA.
Daniel Bryan
DB
Goodman
Harvard-MIT Division of Health Science and Technology, Cambridge, Massachusetts 02139, USA.
David
D
Cox
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.|eGenesis Inc., 1 Kendal Square, Building 200, Cambridge Biolabs, Cambridge, Massachusetts 02139, USA.
Yinan
Y
Kan
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.|eGenesis Inc., 1 Kendal Square, Building 200, Cambridge Biolabs, Cambridge, Massachusetts 02139, USA.
Emal
E
Lesha
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Venkataramanan
V
Soundararajan
Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.|McGovern Institute for Brain Research, MIT, Cambridge, Massachusetts 02139, USA.|Department of Brain and Cognitive Sciences, MIT Cambridge, Cambridge, Massachusetts 02139, USA.
Feng
F
Zhang
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.|Harvard-MIT Division of Health Science and Technology, Cambridge, Massachusetts 02139, USA.|Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts 02115, USA.
George
G
Church
D001483:Base Sequence; D003564:Cytidine Deaminase / Q000235:genetics*; D003641:Deamination; D004926:Escherichia coli / Q000378:metabolism; D000072669:Gene Editing; D005818:Genetic Engineering; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D003564:Cytidine Deaminase