title: Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification.
abstract: Recent studies have shown that zinc finger nucleases (ZFNs) are powerful reagents for making site-specific genomic modifications. The generic structure of these enzymes includes a ZF DNA-binding domain and nuclease domain (Fn) are separated by an amino acid "linker" and cut genomic DNA at sites that have a generic structure (site1)-(spacer)-(site2) where the "spacer" separates the two binding sites. In this work, we compare the activity of ZFNs with different linkers on target sites with different spacer lengths. We found those nucleases with linkers' lengths of 2 or 4 amino acid (aa) efficiently cut at target sites with 5 or 6 base pair (bp) spacers, and that those ZFNs with a 5-aa linker length efficiently cut target sites with 6 or 7 bp spacers. In addition, we demonstrate that the Oligomerized Pool ENgineering (OPEN) platform used for making three-fingered ZF proteins (ZFPs) can be modified to incorporate modular assembly fingers (including those recognizing ANNs, CNNs, and TNNs) and we were able to generate nucleases that efficiently cut cognate target sites. The ability to use module fingers in the OPEN platform at target sites of 5-7 bp spacer lengths increases the probability of finding a ZFN target site to 1 in 4 bp. These findings significantly expand the range of sites that can be potentially targeted by these custom-engineered proteins.Molecular Therapy - Nucleic Acids (2013) 2, e88; doi:10.1038/mtna.2013.13; published online 30 April 2013.
authors: 1] Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA [2] Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
authors: Kimberly A
authors: KA
authors: Wilson
authors: Abbye E
authors: AE
authors: McEwen
authors: Shondra M
authors: SM
authors: Pruett-Miller
authors: Jiuli
authors: J
authors: Zhang
authors: Eric J
authors: EJ
authors: Kildebeck
authors: Matthew H
authors: MH
authors: Porteus
references: Nature. 2005 Jun 2;435(7042):646-51
references: 15806097
references: PLoS Genet. 2009 Feb;5(2):e1000376
references: 19214211
references: J Biol Chem. 2001 Aug 3;276(31):29466-78
references: 11340073
references: Nat Biotechnol. 2002 Feb;20(2):135-41
references: 11821858
references: Nat Biotechnol. 2008 Jul;26(7):808-16
references: 18587387
references: Nat Biotechnol. 2007 Nov;25(11):1298-306
references: 17965707
references: Science. 2003 May 2;300(5620):764
references: 12730594
references: J Biol Chem. 2002 Feb 8;277(6):3850-6
references: 11726671
references: Mol Ther. 2006 Feb;13(2):438-46
references: 16169774
references: Nat Methods. 2008 May;5(5):374-5
references: 18446154
references: J Biol Chem. 2005 Oct 21;280(42):35588-97
references: 16107335
references: Nat Methods. 2010 Feb;7(2):91; author reply 91-2
references: 20111032
references: Mol Cell Biol. 2001 Jan;21(1):289-97
references: 11113203
references: Nat Biotechnol. 2007 Jul;25(7):778-85
references: 17603475
references: Nat Protoc. 2009;4(10):1471-501
references: 19798082
references: Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):2812-7
references: 9501172
references: Mol Cell Biol. 1998 Jan;18(1):93-101
references: 9418857
references: Genetics. 2002 Jul;161(3):1169-75
references: 12136019
references: Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10564-9
references: 9724743
references: Nat Biotechnol. 2007 Jul;25(7):786-93
references: 17603476
references: Nucleic Acids Res. 2000 Sep 1;28(17):3361-9
references: 10954606
references: Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12271-6
references: 14527993
references: Biochemistry. 2003 Feb 25;42(7):2137-48
references: 12590603
references: Genetics. 2006 Apr;172(4):2391-403
references: 16452139
references: Mol Cell. 2008 Jul 25;31(2):294-301
references: 18657511
references: Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1437-41
references: 11171969
references: Nat Methods. 2011 Jan;8(1):67-9
references: 21151135
references: Mol Ther. 2009 Jan;17(1):104-11
references: 19002164
references: Proc Natl Acad Sci U S A. 2008 Apr 15;105(15):5809-14
references: 18359850
references: Science. 2003 May 2;300(5620):763
references: 12730593
references: Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3055-60
references: 17360608
references: Mol Ther. 2005 Oct;12(4):610-7
references: 16039907
references: Nucleic Acids Res. 2005 Oct 26;33(18):5978-90
references: 16251401
references: Mol Ther. 2008 Apr;16(4):707-17
references: 18334988
references: Mol Ther. 2008 Feb;16(2):352-358
references: 28178540