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'''Engineering TALENs'''
'''Engineering TALENs'''


The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. In this case [[artificial gene synthesis]] is problematic because of improper [[annealing]] of the repetitive sequence found in the TALE binding domain.<ref name=Zhang2011/> One solution to this is to use a publicly available software program ([http://helixweb.nih.gov/dnaworks/ DNAWorks]) to calculate [[oligonucleotides]] suitable for assembly in a two step [[PCR]]; oligonucleotide assembly followed by whole gene amplification. A number of modular assembly schemes for generating engineered TALE constructs have also been reported.<ref name=Zhang2011/><ref name = Morbitzer2011>{{Cite doi|10.1093/nar/gkr151}}</ref><ref name = Li2011>{{Cite doi|10.1093/nar/gkr188}}</ref><ref name=Cermak2011>{{Cite doi|10.1093/nar/gkr218}}</ref> Both methods offer a systematic approach to engineering DNA binding domains that is conceptually similar to the modular assembly method for generating [[zinc finger]] DNA recognition domains.
The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. In this case [[artificial gene synthesis]] is problematic because of improper [[annealing]] of the repetitive sequence found in the TALE binding domain.<ref name=Zhang2011/> One solution to this is to use a publicly available software program ([http://helixweb.nih.gov/dnaworks/ DNAWorks]) to calculate [[oligonucleotides]] suitable for assembly in a two step [[PCR]]; oligonucleotide assembly followed by whole gene amplification. A number of modular assembly schemes for generating engineered TALE constructs have also been reported.<ref name=Zhang2011/><ref name = Morbitzer2011>{{Cite doi|10.1093/nar/gkr151}}</ref><ref name = Li2011>{{Cite doi|10.1093/nar/gkr188}}</ref><ref name=Cermak2011>{{Cite doi|10.1093/nar/gkr218}}</ref><ref name=Geissler2011>{{Cite doi|10.1371/journal.pone.0019509 }}</ref><ref name=Weber2011>{{Cite doi|10.1371/journal.pone.0019722 }}</ref> Both methods offer a systematic approach to engineering DNA binding domains that is conceptually similar to the modular assembly method for generating [[zinc finger]] DNA recognition domains.




Revision as of 16:25, 30 May 2011

Transcription Activator-Like Effector Nucleases (TALENs) are artificial restriction enzymes generated by fusing the TAL effector DNA binding domain to a DNA cleavage domain. These reagents enable efficient, programmable, and specific DNA cleavage and represent powerful tools for genome editing in situ. Synthetic transcription factors using TALE domain constructs can also be used for gene regulation by pairing the TALE DNA binding domain with an endogenous activation domain affecting expression at specific sites in complex genomes.[1][2][3] Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any DNA sequence.[4]


DNA binding domain

Transcription activator-like effectors are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a highly conserved 33-34 amino acid sequence with the exception of the 12th and 13th amino acids. These two locations are highly variable (Repeat Variable Diresidue) and show a strong correlation with specific nucleotide recognition.[5][6] This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.[7]


DNA cleavage domain

The non-specific DNA cleavage domain from the end of the FokI endonuclease can be used to construct hybrid nucleases that are active in a yeast assay.[7][8] These reagents are also active in plant cells[9] and in animal cells.[2] The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing.


Engineering TALENs

The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. In this case artificial gene synthesis is problematic because of improper annealing of the repetitive sequence found in the TALE binding domain.[3] One solution to this is to use a publicly available software program (DNAWorks) to calculate oligonucleotides suitable for assembly in a two step PCR; oligonucleotide assembly followed by whole gene amplification. A number of modular assembly schemes for generating engineered TALE constructs have also been reported.[3][10][11][12][13][14] Both methods offer a systematic approach to engineering DNA binding domains that is conceptually similar to the modular assembly method for generating zinc finger DNA recognition domains.


Workflow

TALEN copy


Transfection

Once the TALEN genes have been assembled they are inserted into plasmids; the plasmids are then used to transfect the target cell where the gene products are expressed and enter the nucleus to access the genome.


Genome Editing

TALENs can be used to edit genomes by inducing double strand breaks (DSB) which cells respond to with repair mechanisms. Non-homologous end joining (NHEJ) reconnects DNA from either side of a double strand break where there is very little or no sequence overlap for annealing. This repair mechanism induces errors in the genome via insertion, deletion, or chromosomal rearrangement; any of which may leave the gene products coded at that location non-functional.[2] Additionally, DNA can be introduced into a genome through NHEJ in the presence of exogenous double stranded DNA fragments.[2] Homology directed repair can also introduce foreign DNA at the DSB as the transfected double stranded sequences are used as template for the repair enzymes.[2]

See also

References

  1. ^ Attention: This template ({{cite doi}}) is deprecated. To cite the publication identified by doi: 10.1073/pnas.1013133107 , please use {{cite journal}} (if it was published in a bona fide academic journal, otherwise {{cite report}} with |doi= 10.1073/pnas.1013133107 instead.
  2. ^ a b c d e Miller, Jeffrey (2011). "A TALE nuclease architecture for efficient genome editing". Nature Biotechnology. 29 (2): 143–148. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help); Unknown parameter |month= ignored (help)
  3. ^ a b c Zhang, Feng (2011). "Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription". Nature Biotechnology. 29 (2): 149–153. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help); Unknown parameter |month= ignored (help)
  4. ^ Boch, Jens (2011). "TALEs of genome targeting". Nature Biotechnology. 29 (2): 135–136. {{cite journal}}: Unknown parameter |month= ignored (help)
  5. ^ Boch, Jens (2009). "Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors". Science. 326: 1509–1512. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help); Unknown parameter |month= ignored (help)
  6. ^ Moscou, Matthew J. (2009). "A Simple Cipher Governs DNA Recognition by TAL Effectors". Science. 326: 1501. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help); Unknown parameter |month= ignored (help)
  7. ^ a b Christian, Michelle (2010). "Targeting DNA Double-Strand Breaks with TAL Effector Nucleases". Genetics. 186: 757–761. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help); Unknown parameter |month= ignored (help)
  8. ^ Li, Ting (2010). "TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain". Nucleic Acids Research: 1–14. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help); Unknown parameter |month= ignored (help)
  9. ^ Mahfouz, Magdy M. (2010). "De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-stran breaks". PNAS. 108 (6): 2623–2628. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help); Unknown parameter |month= ignored (help)
  10. ^ Attention: This template ({{cite doi}}) is deprecated. To cite the publication identified by doi:10.1093/nar/gkr151, please use {{cite journal}} (if it was published in a bona fide academic journal, otherwise {{cite report}} with |doi=10.1093/nar/gkr151 instead.
  11. ^ Attention: This template ({{cite doi}}) is deprecated. To cite the publication identified by doi:10.1093/nar/gkr188, please use {{cite journal}} (if it was published in a bona fide academic journal, otherwise {{cite report}} with |doi=10.1093/nar/gkr188 instead.
  12. ^ Attention: This template ({{cite doi}}) is deprecated. To cite the publication identified by doi:10.1093/nar/gkr218, please use {{cite journal}} (if it was published in a bona fide academic journal, otherwise {{cite report}} with |doi=10.1093/nar/gkr218 instead.
  13. ^ Attention: This template ({{cite doi}}) is deprecated. To cite the publication identified by doi:10.1371/journal.pone.0019509 , please use {{cite journal}} (if it was published in a bona fide academic journal, otherwise {{cite report}} with |doi=10.1371/journal.pone.0019509 instead.
  14. ^ Attention: This template ({{cite doi}}) is deprecated. To cite the publication identified by doi:10.1371/journal.pone.0019722 , please use {{cite journal}} (if it was published in a bona fide academic journal, otherwise {{cite report}} with |doi=10.1371/journal.pone.0019722 instead.

External links

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