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Generation of knockout rabbits using transcription activator-like effector nucleases.

Wang Y, Fan N, Song J, Zhong J, Guo X, Tian W, Zhang Q, Cui F, Li L, Newsome PN, Frampton J, Esteban MA, Lai L - Cell Regen (Lond) (2014)

Bottom Line: Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research.They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci.This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, 510530 China.

ABSTRACT
Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

No MeSH data available.


Related in: MedlinePlus

Schematic depiction of how ZFNs and TALENs recognize target DNA and induce genome editing. ZFP stands for zinc-finger protein, NLS for nuclear localization signal, N-terminal and C-terminal for amino- and carboxyl-terminal, respectively. DSBs induced by the designer nucleases can be repaired by homology-directed repair or nonhomologous end joining, which can result in knock-ins or knockouts, respectively. FOKI can be substituted by other restriction endonucleases [22].
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Fig1: Schematic depiction of how ZFNs and TALENs recognize target DNA and induce genome editing. ZFP stands for zinc-finger protein, NLS for nuclear localization signal, N-terminal and C-terminal for amino- and carboxyl-terminal, respectively. DSBs induced by the designer nucleases can be repaired by homology-directed repair or nonhomologous end joining, which can result in knock-ins or knockouts, respectively. FOKI can be substituted by other restriction endonucleases [22].

Mentions: To overcome the above-mentioned issues, a new technology termed “genome editing” has emerged that allows investigators to modify virtually any gene in a variety of organisms and cell types [18]. Two remarkable examples of this novel approach are zinc-finger nucleases (ZFNs) and transcription activator-like effector (TALE) nucleases (TALENs). These 2 types of designer nucleases are composed of a programmable module that can be adapted to recognize specific genomic sequences, and a non-specific DNA cleavage domain (Figure 1). This combination can produce DNA double-strand breaks (DSBs) at specific loci, which by means of error-prone nonhomologous end joining or homology-directed repair can result in knockouts, nucleotide substitutions, knock-ins, and even larger chromosomal rearrangements [18]. The zinc-finger domain is one of the most frequent motifs in mammalian DNA-binding proteins. Its modular (ββα) structure exposes several amino acids that recognize 3 base pairs in the major groove of DNA [19]. Such unique mode of action made attractive the design of multimodular custom-made DNA-binding proteins with site-specific affinities, which were then fused to the restriction endonuclease FokI [19] and pioneered the field. On the other hand, TALE proteins are naturally occurring proteins from the plant pathogen Xanthomonas (a type of proteobacteria) that contain individual repeats (each typically consisting of 34 amino acids) targeting each a single DNA base pair [20]. Like with ZFNs, TALE repeats can be assembled into a multimodular protein that recognizes contiguous DNA sequences (Figure 1). Yet, the single base recognition by TALE repeats makes the design of TALENs more flexible than ZFNs. In fact, a series of systematized strategies have been developed that enable relatively quick and affordable design/assembly compared to the more tedious and costly ZFNs [18].Figure 1


Generation of knockout rabbits using transcription activator-like effector nucleases.

Wang Y, Fan N, Song J, Zhong J, Guo X, Tian W, Zhang Q, Cui F, Li L, Newsome PN, Frampton J, Esteban MA, Lai L - Cell Regen (Lond) (2014)

Schematic depiction of how ZFNs and TALENs recognize target DNA and induce genome editing. ZFP stands for zinc-finger protein, NLS for nuclear localization signal, N-terminal and C-terminal for amino- and carboxyl-terminal, respectively. DSBs induced by the designer nucleases can be repaired by homology-directed repair or nonhomologous end joining, which can result in knock-ins or knockouts, respectively. FOKI can be substituted by other restriction endonucleases [22].
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230510&req=5

Fig1: Schematic depiction of how ZFNs and TALENs recognize target DNA and induce genome editing. ZFP stands for zinc-finger protein, NLS for nuclear localization signal, N-terminal and C-terminal for amino- and carboxyl-terminal, respectively. DSBs induced by the designer nucleases can be repaired by homology-directed repair or nonhomologous end joining, which can result in knock-ins or knockouts, respectively. FOKI can be substituted by other restriction endonucleases [22].
Mentions: To overcome the above-mentioned issues, a new technology termed “genome editing” has emerged that allows investigators to modify virtually any gene in a variety of organisms and cell types [18]. Two remarkable examples of this novel approach are zinc-finger nucleases (ZFNs) and transcription activator-like effector (TALE) nucleases (TALENs). These 2 types of designer nucleases are composed of a programmable module that can be adapted to recognize specific genomic sequences, and a non-specific DNA cleavage domain (Figure 1). This combination can produce DNA double-strand breaks (DSBs) at specific loci, which by means of error-prone nonhomologous end joining or homology-directed repair can result in knockouts, nucleotide substitutions, knock-ins, and even larger chromosomal rearrangements [18]. The zinc-finger domain is one of the most frequent motifs in mammalian DNA-binding proteins. Its modular (ββα) structure exposes several amino acids that recognize 3 base pairs in the major groove of DNA [19]. Such unique mode of action made attractive the design of multimodular custom-made DNA-binding proteins with site-specific affinities, which were then fused to the restriction endonuclease FokI [19] and pioneered the field. On the other hand, TALE proteins are naturally occurring proteins from the plant pathogen Xanthomonas (a type of proteobacteria) that contain individual repeats (each typically consisting of 34 amino acids) targeting each a single DNA base pair [20]. Like with ZFNs, TALE repeats can be assembled into a multimodular protein that recognizes contiguous DNA sequences (Figure 1). Yet, the single base recognition by TALE repeats makes the design of TALENs more flexible than ZFNs. In fact, a series of systematized strategies have been developed that enable relatively quick and affordable design/assembly compared to the more tedious and costly ZFNs [18].Figure 1

Bottom Line: Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research.They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci.This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology of the Chinese Academy of Sciences and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, 510530 China.

ABSTRACT
Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

No MeSH data available.


Related in: MedlinePlus