Transcription Activator-Like Effector Nuclease
Encyclopedia
Transcription Activator-Like Effector Nucleases (TALENs) are artificial restriction enzyme
s generated by fusing the TAL effector
DNA binding domain to a DNA cleavage domain.
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.
Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any DNA sequence.
s 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. 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.
endonuclease can be used to construct hybrid nuclease
s that are active in a yeast assay. These reagents are also active in plant cells
and in animal cells. Initial TALEN studies used the wild-type FokI cleavage domain, but some subsequent TALEN studies also used FokI cleavage domain variants with mutations designed to improve cleavage specificity and cleavage activity. 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. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity.
of the repetitive sequence found in the TALE binding domain. 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. 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.
s; the plasmids are then used to transfect the target cell where the gene products are expressed and enter the nucleus to access the genome.
(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 such errors may render the gene products coded at that location non-functional. Additionally, DNA can be introduced into a genome through NHEJ in the presence of exogenous double-stranded DNA fragments. Homology directed repair
can also introduce foreign DNA at the DSB as the transfected double-stranded sequences are used as templates for the repair enzymes. TALENs have been used to generate stably modified human embryonic stem cell
and induced pluripotent stem cell
(IPSCs) clones, to generate knockout C. elegans
, knockout rat
s, and knockout zebrafish.
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
s generated by fusing the TAL effector
TAL effector
TAL effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when they infect various plant species...
DNA binding domain to a DNA cleavage domain.
Use
These reagents enable efficient, programmable, and specific DNA cleavage and represent powerful tools for genomeGenome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
editing in situ
In situ
In situ is a Latin phrase which translated literally as 'In position'. It is used in many different contexts.-Aerospace:In the aerospace industry, equipment on board aircraft must be tested in situ, or in place, to confirm everything functions properly as a system. Individually, each piece may...
. Synthetic transcription factors using TALE domain constructs can also be used for gene regulation by pairing the TALE DNA binding domain with an endogenous
Endogenous
Endogenous substances are those that originate from within an organism, tissue, or cell. Endogenous retroviruses are caused by ancient infections of germ cells in humans, mammals and other vertebrates...
activation domain affecting expression at specific sites in complex genomes.
Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any DNA sequence.
DNA binding domain
TAL effectorTAL effector
TAL effectors are proteins secreted by Xanthomonas bacteria via their type III secretion system when they infect various plant species...
s are proteins secreted by Xanthomonas
Xanthomonas
Xanthomonas is a genus of Proteobacteria, many of which cause plant diseases. Most varieties of Xanthomonas are available from the National Collection of Plant Pathogenic Bacteria in the United Kingdom and other international culture collections such as ICMP in New Zealand, CFBP in France, and...
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
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
recognition. 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.
DNA cleavage domain
The non-specific DNA cleavage domain from the end of the FokIFokI
The enzyme FokI, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal...
endonuclease can be used to construct hybrid nuclease
Nuclease
A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Older publications may use terms such as "polynucleotidase" or "nucleodepolymerase"....
s that are active in a yeast assay. These reagents are also active in plant cells
and in animal cells. Initial TALEN studies used the wild-type FokI cleavage domain, but some subsequent TALEN studies also used FokI cleavage domain variants with mutations designed to improve cleavage specificity and cleavage activity. 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. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity.
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 annealingAnnealing
Annealing may refer to:*Annealing , a heat treatment that alters the microstructure of a material causing changes in properties such as strength and hardness and ductility*Annealing , heating a piece of glass to remove stress...
of the repetitive sequence found in the TALE binding domain. 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. Both methods offer a systematic approach to engineering DNA binding domains that is conceptually similar to the modular assembly method for generating zinc finger
Zinc finger
Zinc fingers are small protein structural motifs that can coordinate one or more zinc ions to help stabilize their folds. They can be classified into several different structural families and typically function as interaction modules that bind DNA, RNA, proteins, or small molecules...
DNA recognition domains.
Transfection
Once the TALEN genes have been assembled they are inserted into plasmidPlasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
s; 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 joiningNon-homologous end joining
Non-homologous end joining is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homologous recombination, which requires a homologous sequence to guide...
(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 such errors may render the gene products coded at that location non-functional. Additionally, DNA can be introduced into a genome through NHEJ in the presence of exogenous double-stranded DNA fragments. Homology directed repair
Homologous recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...
can also introduce foreign DNA at the DSB as the transfected double-stranded sequences are used as templates for the repair enzymes. TALENs have been used to generate stably modified human embryonic stem cell
Embryonic stem cell
Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells...
and induced pluripotent stem cell
Induced pluripotent stem cell
Induced pluripotent stem cells, commonly abbreviated as iPS cells or iPSCs are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing a "forced" expression of specific genes....
(IPSCs) clones, to generate knockout C. elegans
Caenorhabditis elegans
Caenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
, knockout rat
Knockout rat
A knockout rat is a genetically engineered rat with a single gene turned off through a targeted mutation used for academic and pharmaceutical research. Knockout rats can mimic human diseases and are important tools for studying gene function and for drug discovery and development...
s, and knockout zebrafish.
External links
- www.taleffectors.com An open resource for TAL effector constructs
- Cellectis bioresearch A commercial supplier of TALENs