title: The I-TevI nuclease and linker domains contribute to the specificity of monomeric TALENs.
abstract: Precise genome editing in complex genomes is enabled by engineered nucleases that can be programmed to cleave in a site-specific manner. Here, we fused the small, sequence-tolerant monomeric nuclease domain from the homing endonuclease I-TevI to transcription-like activator effectors (TALEs) to create monomeric Tev-TALE nucleases (Tev-mTALENs). Using the PthXo1 TALE scaffold to optimize the Tev-mTALEN architecture, we found that choice of the N-terminal fusion point on the TALE greatly influenced activity in yeast-based assays, and that the length of the linker used affected the optimal spacing of the TALE binding site from the I-TevI cleavage site, specified by the motif 5'-CNNNG-3'. By assaying activity on all 64 possible sequence variants of this motif, we discovered that in the Tev-mTALEN context, I-TevI prefers A/T-rich triplets over G/C-rich ones at the cleavage site. Profiling of nucleotide requirements in the DNA spacer that separates the CNNNG motif from the TALE binding site revealed substantial, but not complete, tolerance to sequence variation. Tev-mTALENs showed robust mutagenic activity on an episomal target in HEK 293T cells consistent with specific cleavage followed by nonhomologous end-joining repair. Our data substantiate the applicability of Tev-mTALENs as genome-editing tools but highlight DNA spacer and cleavage site nucleotide preferences that, while enhancing specificity, do confer moderate targeting constraints.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada N6A 5C1.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Kleinstiver
authors.affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University, 334 Plant Science, Ithaca, New York 14853.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada N6A 5C1.
authors.forename: Jason M
authors.initials: JM
authors.lastname: Wolfs
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada N6A 5C1.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Kolaczyk
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada N6A 5C1.
authors.forename: Brendon
authors.initials: B
authors.lastname: McDowell
authors.affiliation: Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada N6A 5B7.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada N6A 5C1 Robarts Research Institute, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada N6A 5B7.
authors.forename: Caroline
authors.initials: C
authors.lastname: Schild-Poulter
authors.affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University, 334 Plant Science, Ithaca, New York 14853 dedgell@uwo.ca ajb7@cornell.edu.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Bogdanove
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada N6A 5C1 dedgell@uwo.ca ajb7@cornell.edu.
authors.forename: David R
authors.initials: DR
authors.lastname: Edgell
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D018390:Gene Targeting; D014644:Genetic Variation; D005822:Genetic Vectors; D018398:Homeodomain Proteins; D006801:Humans; D008969:Molecular Sequence Data; D059372:Nucleotide Motifs; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins; D013379:Substrate Specificity
chemical_list: D018398:Homeodomain Proteins; D011993:Recombinant Fusion Proteins; D004706:Endodeoxyribonucleases; C065074:I-TEVI endonuclease
keywords: GIY-YIG nuclease; I-TevI; TAL effector; genome editing; monomeric TALEN
references.citation: Science. 2009 Dec 11;326(5959):1509-12
references.pmid: 19933107
references.citation: Plant Mol Biol. 2012 Feb;78(3):311-21
references.pmid: 22167390
references.citation: Nucleic Acids Res. 2012 Mar;40(6):2623-38
references.pmid: 22135304
references.citation: Science. 2009 Dec 11;326(5959):1501
references.pmid: 19933106
references.citation: Science. 2003 May 2;300(5620):764
references.pmid: 12730594
references.citation: Proc Natl Acad Sci U S A. 2012 May 22;109(21):8061-6
references.pmid: 22566637
references.citation: J Mol Biol. 1997 Feb 7;265(5):494-506
references.pmid: 9048944
references.citation: Science. 2013 Feb 15;339(6121):823-6
references.pmid: 23287722
references.citation: Nat Methods. 2011 Aug 07;8(9):765-70
references.pmid: 21822273
references.citation: Science. 2014 Jan 3;343(6166):84-87
references.pmid: 24336571
references.citation: Nature. 2009 May 21;459(7245):442-5
references.pmid: 19404258
references.citation: Nat Biotechnol. 2011 Feb;29(2):143-8
references.pmid: 21179091
references.citation: Genetics. 2010 Oct;186(2):757-61
references.pmid: 20660643
references.citation: Nat Biotechnol. 2012 May;30(5):460-5
references.pmid: 22484455
references.citation: Biochem Soc Trans. 2011 Apr;39(2):584-8
references.pmid: 21428944
references.citation: Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8554-61
references.pmid: 12077294
references.citation: EMBO J. 1993 May;12(5):2141-9
references.pmid: 8491202
references.citation: Science. 2013 Aug 23;341(6148):833-6
references.pmid: 23970676
references.citation: Gene. 1997 Dec 5;203(1):43-9
references.pmid: 9426005
references.citation: Yeast. 2007 Oct;24(10):913-9
references.pmid: 17583893
references.citation: Proc Natl Acad Sci U S A. 1992 May 15;89(10):4275-9
references.pmid: 1584761
references.citation: Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10570-5
references.pmid: 9724744
references.citation: Nat Biotechnol. 2007 Jul;25(7):786-93
references.pmid: 17603476
references.citation: Nucleic Acids Res. 2000 Sep 1;28(17):3361-9
references.pmid: 10954606
references.citation: Biochemistry. 2011 Jun 7;50(22):5033-41
references.pmid: 21528840
references.citation: J Mol Biol. 2004 Nov 5;343(5):1231-41
references.pmid: 15491609
references.citation: Nat Biotechnol. 2011 Aug 07;29(9):816-23
references.pmid: 21822255
references.citation: Science. 2011 Sep 30;333(6051):1843-6
references.pmid: 21960622
references.citation: EMBO J. 2001 Jul 16;20(14):3631-7
references.pmid: 11447104
references.citation: Nucleic Acids Res. 1990 Oct 25;18(20):6097-100
references.pmid: 2172928
references.citation: Nucleic Acids Res. 2011 Aug;39(14):6315-25
references.pmid: 21459844
references.citation: Nat Biotechnol. 2014 Mar;32(3):279-284
references.pmid: 24463574
references.citation: Proc Natl Acad Sci U S A. 2011 Aug 9;108(32):13077-82
references.pmid: 21784983
references.citation: Nat Commun. 2013;4:1762
references.pmid: 23612303
references.citation: J Mol Biol. 1995 Mar 24;247(2):197-210
references.pmid: 7707369
references.citation: Nat Biotechnol. 2013 Sep;31(9):833-8
references.pmid: 23907171
references.citation: Mol Biosyst. 2014 Mar 4;10(3):446-53
references.pmid: 24336919
references.citation: Science. 2013 Feb 15;339(6121):819-23
references.pmid: 23287718
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 5;103(27):10503-10508
references.pmid: 16798873
references.citation: Science. 2003 May 2;300(5620):763
references.pmid: 12730593
references.citation: Nat Biotechnol. 2011 Jul 07;29(8):731-4
references.pmid: 21738127
references.citation: Nat Rev Genet. 2013 Feb;14(2):80
references.pmid: 23322222
references.citation: J Mol Biol. 2011 Jan 21;405(3):630-41
references.pmid: 21094162