Sage Journals: Discover world-class research

Abstract

Engineered zinc finger nucleases (ZFNs) are a tool for genome manipulation that are of great interest to scientists in many fields. To meet the needs of researchers wishing to employ ZFNs, an inexpensive, rapid assembly procedure would be beneficial to laboratories that do not have access to the proprietary reagents often required for ZFN production. Using freely available sequence data derived from the Zinc Finger Targeter database, we developed a protocol for synthesis and directed insertion of user-defined ZFNs into a versatile plasmid expression system. This oligonucleotide-based isothermal DNA assembly protocol was used to determine whether we could generate functional nucleases capable of endogenous gene editing. We targeted the human α-l-iduronidase (IDUA) gene on chromosome 4, mutations of which result in the severe lysosomal storage disease mucopolysaccharidosis type I. In approximately 1 week we were able to design, assemble, and test six IDUA-specific ZFNs. In a single-stranded annealing assay five of the six candidates we tested performed at a level comparable to or surpassing previously reported ZFNs. One of the five subsequently showed nuclease activity at the endogenous genomic IDUA locus. To our knowledge, this is the first demonstration of in silico-designed, oligonucleotide-assembled, synthetic ZFNs, requiring no specialized templates or reagents that are capable of endogenous human gene target site activity. This method, termed CoDA-syn (context-dependent assembly-synthetic), should facilitate a more widespread use of ZFNs in the research community.

Get full access to this article

View all access options for this article.

References

Alwin

, Gere

M.B.

, Guhl

et al. 2005. Custom zinc-finger nucleases for use in human cells. Mol. Ther., 12:610–617.

Bhakta

M.S.

, Segal

D.J.

2010. The generation of zinc finger proteins by modular assembly. Methods Mol. Biol., 649:3–30.

Carroll

, Morton

J.J.

, Beumer

K.J.

, Segal

D.J.

2006. Design, construction and in vitro testing of zinc finger nucleases. Nat. Protoc., 1:1329–1341.

Cermak

, Doyle

E.L.

, Christian

et al. 2011. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res., 39:e82.

Christian

, Cermak

, Doyle

E.L.

et al. 2010. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics, 186:757–761.

Cleary

M.A.

, Wraith

J.E.

1995. The presenting features of mucopolysaccharidosis type IH (Hurler syndrome) Acta Paediatr., 84:337–339.

Doyon

, Vo

T.D.

, Mendel

M.C.

et al. 2011. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat. Methods, 8:74–79.

Gibson

D.G.

, Smith

H.O.

, Hutchison

C.A.

III

et al. 2011. Chemical synthesis of the mouse mitochondrial genome. Nat. Methods, 7:901–903.

Gibson

D.G.

, Young

, Chuang

R.Y.

et al. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods, 6:343–345.

10.

Guschin

D.Y.

, Waite

A.J.

, Katibah

G.E.

et al. 2010. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol., 649:247–256.

11.

Handel

E.M.

, Alwin

, Cathomen

2009. Expanding or restricting the target site repertoire of zinc-finger nucleases: The inter-domain linker as a major determinant of target site selectivity. Mol. Ther., 17:104–111.

12.

Kim

H.J.

, Lee

H.J.

, Kim

et al. 2009. Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res., 19:1279–1288.

13.

Kim

, Lee

M.J.

, Kim

et al. 2011. Preassembled zinc-finger arrays for rapid construction of ZFNs. Nat. Methods, 8:7.

14.

Maeder

M.L.

, Thibodeau-Beganny

, Osiak

et al. 2008. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol. Cell, 31:294–301.

15.

Meng

, Noyes

M.B.

, Zhu

L.J.

et al. 2008. Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol., 26:695–701.

16.

Miller

J.C.

, Holmes

M.C.

, Wang

et al. 2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol., 25:778–785.

17.

Miller

J.C.

, Tan

, Qiao

et al. 2011. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol., 29:143–148.

18.

Muenzer

, Wraith

J.E.

, Clarke

L.A.

2009. Mucopolysaccharidosis I: Management and treatment guidelines. Pediatrics, 123:19–29.

19.

Orlando

S.J.

, Santiago

, Dekelver

R.C.

et al. 2010. Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res., 38:e152.

20.

Osborn

M.J.

, Panoskaltsis-Mortari

, McElmurry

R.T.

et al. 2005. A picornaviral 2A-like sequence-based tricistronic vector allowing for high-level therapeutic gene expression coupled to a dual-reporter system. Mol. Ther., 12:569–574.

21.

Porteus

2010. Creating zinc finger nucleases using a modular-assembly approach. Cold Spring Harbor Protoc., 2010pdb.prot5530.

22.

Porteus

M.H.

, Carroll

2005. Gene targeting using zinc finger nucleases. Nat. Biotechnol., 23:967–973.

23.

Pruett-Miller

S.M.

, Connelly

J.P.

, Maeder

M.L.

et al. 2008. Comparison of zinc finger nucleases for use in gene targeting in mammalian cells. Mol. Ther., 16:707–717.

24.

Ramirez

C.L.

, Foley

J.E.

, Wright

D.A.

et al. 2008. Unexpected failure rates for modular assembly of engineered zinc fingers. Nat. Methods, 5:374–375.

25.

Rasband

W.S.

1997–2011. ImageJ. U.S. National Institutes of Health: Bethesda, MD http://imagej.nih.gov/ij/. 2011 June .

26.

Remy

, Tesson

, Menoret

et al. 2010. Zinc-finger nucleases: A powerful tool for genetic engineering of animals. Transgenic Res., 19:363–371.

27.

Sander

J.D.

, Dahlborg

E.J.

, Goodwin

M.J.

et al. 2011. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA) Nat. Methods, 8:67–69.

28.

Sander

J.D.

, Zaback

, Joung

J.K.

et al. 2007. Zinc Finger Targeter (ZiFiT): An engineered zinc finger/target site design tool. Nucleic Acids Res., 35:W599–W605.

29.

Voskarides

, Deltas

2009. Screening for mutations in kidney-related genes using SURVEYOR nuclease for cleavage at heteroduplex mismatches. J. Mol. Diagn., 11:311–318.

30.

Xiong

A.S.

, Peng

R.H.

, Zhuang

et al. 2008. Chemical gene synthesis: Strategies, softwares, error corrections, and applications. FEMS Microbiol. Rev., 32:522–540.

31.

Zhang

, Maeder

M.L.

, Unger-Wallace

et al. 2010. High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proc. Natl. Acad. Sci. U.S.A., 107:12028–12033.

32.

Zou

, Maeder

M.L.

, Mali

et al. 2009. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell, 5:97–110.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.14 MB

Synthetic Zinc Finger Nuclease Design and Rapid Assembly

Abstract

Get full access to this article

References

Supplementary Material