De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks.
Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.
Center for Plant Stress Genomics and Technology, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia.
Magdy M
MM
Mahfouz
Lixin
L
Li
Md
M
Shamimuzzaman
Anjar
A
Wibowo
Xiaoyun
X
Fang
Jian-Kang
JK
Zhu
D004247:DNA / Q000737:chemistry*; D053903:DNA Breaks, Double-Stranded; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D004247:DNA; D003851:Deoxyribonucleases
Nat Biotechnol. 2005 Aug;23(8):967-73
16082368
Genetics. 2010 Oct;186(2):757-61
20660643
New Phytol. 2010 Sep;187(4):1048-1057
20345643
Science. 2009 Dec 11;326(5959):1501
19933106
Mol Plant Microbe Interact. 2005 Aug;18(8):838-48
16134896
Annu Rev Phytopathol. 2010;48:419-36
19400638
Mol Cell. 2008 Jul 25;31(2):294-301
18657511
Mol Ther. 2008 Jul;16(7):1200-7
18545224
Nucleic Acids Res. 2011 Jan;39(1):359-72
20699274
Gene Ther. 2008 Nov;15(22):1463-8
18784746
Science. 2009 Dec 11;326(5959):1509-12
19933107
Nat Protoc. 2006;1(3):1637-52
17406455
J Mol Biol. 2001 May 25;309(1):69-78
11491302
Science. 2009 Dec 11;326(5959):1491-2
20007890
Nat Rev Genet. 2010 Sep;11(9):636-46
20717154
Nature. 2009 May 21;459(7245):442-5
19404258
Biol Chem. 1998 Apr-May;379(4-5):489-95
9628342
Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21617-22
21106758
Trends Plant Sci. 2010 Jun;15(6):308-21
20347379
Gene. 1997 Dec 5;203(1):43-9
9426005
Plant J. 2005 Apr;42(2):175-87
15807781
Plant Physiol. 2007 Dec;145(4):1144-54
18056864
Nat Protoc. 2006;1(3):1329-41
17406419
Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10570-5
9724744
Nat Biotechnol. 2007 Jul;25(7):786-93
17603476
Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20526-31
19910532
Mol Ther. 2009 Jan;17(1):104-11
19002164
Science. 2010 Feb 12;327(5967):802-3
20150481
Nat Methods. 2008 May;5(5):374-5
18446154
Methods Mol Biol. 2008;435:63-77
18370068
Nat Biotechnol. 2008 Jun;26(6):695-701
18500337
Mol Gen Genet. 1993 Jan;236(2-3):331-40
8437577
Mol Cell Biol. 2001 Jan;21(1):289-97
11113203
Proc Natl Acad Sci U S A. 2010 Jun 29;107(26):12028-33
20508152
Plant J. 2005 Nov;44(4):693-705
16262717