Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Aug;39(14):6315-25.
doi: 10.1093/nar/gkr188. Epub 2011 Mar 31.

Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

Ting Li  1 , Sheng Huang, Xuefeng Zhao, David A Wright, Susan Carpenter, Martin H Spalding, Donald P Weeks, Bing Yang
Affiliations

Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

Ting Li et al. Nucleic Acids Res. 2011 Aug.

Abstract

Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
DNA alterations by natural TAL effector-derived TALENs at chromosomally integrated paired EBE target sites in the URA3 gene. (A) Schematic of TALENs used in the study. Full-length TALEs were fused with the homodimeric cleavage domain of FokI (FN). The number of amino acids in the separate domains is shown above each region (NLS, nuclear localization motifs; AD, transcription activation domain). (B) Target sequences between the first and second codons of the yeast URA3 gene targeted for cleavage by hybrid TALENs derived from AvrXa7 and PthXo1. (C) Alignment of genomic sequences of mutants and their parental strain at the TALEN target site. The number of nucleotides inserted (lowercase letters in red) or deleted (dashes) compared to parental sequences (PT) from each mutant colony is indicated on the right of each sequence.
Figure 2.
Figure 2.
Design and modular construction of dTALENs. (A) TALEN repeat gene sets for modular construction of multi-repeat TALEN genes. Each set contains four single-repeat genes each encoding one of the four ‘core’ TALEN repeat modules containing NI, NG, NN and HD RVDs with binding specificity for A, T, G and C nucleotides, respectively. The core repeats in each set (boxed) contains a 5′- and 3′-termini unique to that particular set—as listed in the inset table. For construction of a TALEN repeat array recognizing a specific 8 nt DNA recognition sequence (e.g. AGGTACTC), an NI repeat gene is selected from Set 1, an NN repeat gene from Set 2, an NN repeat gene from Set 3, an NG repeat gene from Set 4, etc. The 5′- and 3′-terminal regions are designed such that BsmBI digestion results in generation of 5′ and 3′ 4-base overhangs. Because of the unique complementarity of 3′ overhangs from Set 1 genes with the 5′ overhang of genes from Set 2, the complementarity of the 3′ overhangs of Set 2 with 5′ overhangs from Set 3, etc., the annealing and ligation of overhangs results in one, and only one, ordered alignment of the eight repeat genes that, when translated, will specifically recognize and bind (in the present example) the AGGTACTC DNA recognition sequence. (B) Once two (or three) such blocks of repeats are constructed, they are combined in a similar ordered fashion to create a TALE repeat region that is capable of binding a specific 16 (or 24) bp target site. (C) The assembled TALE repeat domains are cloned into the TALEN repeat-deficient pAvrXa7-FN scaffold to create a candidate dTALEN.
Figure 3.
Figure 3.
Yeast SSA assay of modularly assembled dTALENs targeting three endogenous yeast genes. (A) RVD sequences within repeat modules of 10 custom-synthesized dTALENs. N* represents dTALEN repeat modules with the 13th amino acid missing. (B) Schematic of the yeast SSA assay for measuring dTALEN activity based on plasmid-borne HR. Individual candidate dTALENs are assayed in combination with AvrXa7-FN for their ability to stimulate the recombination between the duplicated regions of LacZ gene (hatched boxes), leading to formation of a functional lacZ gene. (C) Activities of individual dTALENs in creating DSBs as detected in a β-galactosidase assay. Control denotes the β-galactosidase activity (<5 U) of yeast cells lacking a functional TALEN gene. Error bars denote SD; n = 3.
Figure 4.
Figure 4.
dTALEN-induced gene modifications by NHEJ and HR. (A) The frequency of gene disruption induced by five sets of paired dTALENs at five specific gene target sites as measured by the numbers of colonies with the indicated mutant phenotypes. ‘—’ denotes not applicable. (B) TALEN-induced insertion/deletion mutations at three of five gene loci tested. (Mutations at the other two target sites are provided in Supplementary Figure S4.) Genomic sequences from each mutant clone at the relevant loci are aligned with the respective wild-type sequences. dTALEN target sites are underlined. The number of nucleotides inserted (bold uppercase letters) or deleted (dashes) is indicated to the right of each sequence. (C) TALEN-induced HR as measured by the percentage of yeast colonies displaying the indicated phenotypes.
Figure 5.
Figure 5.
Growth of yeast cells expressing the paired TALEN and paired ZFN genes. Strains containing plasmids encoding the indicated nuclease genes (on the left side of each row) or lack thereof (pCP3M and pCP4M) were serially diluted (from 104 to 10 cells), applied as spots to SC medium plates lacking histidine (for pCP3M and its derived plasmids) and leucine (for pCP4M and its derived plasmids) and allowed to grow for 4 days.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cathomen T, Joung JK. Zinc-finger nucleases: The next generation emerges. Mol. Ther. 2008;16:1200–1207. - PubMed
    1. Carroll D. Progress and prospects: Zinc-finger nucleases as gene therapy agents. Gene Ther. 2008;15:1463–1468. - PMC - PubMed
    1. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 2010;11:636–646. - PubMed
    1. Jasin M. Genetic manipulation of genomes with rare-cutting endonucleases. Trends Genet. 1996;12:224–228. - PubMed
    1. Vasquez KM, Marburger K, Intody Z, Wilson JH. Manipulating the mammalian genome by homologous recombination. Proc. Natl Acad. Sci. USA. 2001;98:8403–8410. - PMC - PubMed

Publication types

MeSH terms

Substances