gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
Regular Plasmid TALEN Vector
Transcription activator-like effector nuclease (TALEN) knockout vectors are among several types of emerging genome editing tools that can quickly and efficiently create mutations at target sites of a genome (the other two popular ones being CRISPR/Cas9 and ZFN). These plasmid vectors encode artificial restriction enzymes known as TALENs, which consist of an engineered Transcription Activator-Like (TAL) DNA-binding domain fused to a nuclease domain. Our TALEN knockout vectors utilize the Fokl nuclease domain (from Flavobacterium okeanokoites) linked to a TAL domain designed to bind a target sequence specified by the user.
TALs are a class of DNA-binding proteins naturally found in the plant pathogen Xanthomonas sp. A TAL molecule contains a DNA-binding domain which consists of tandem 34aa modules, each of which specifies binding to a specific DNA base. Almost any arbitrary 18-22 bp DNA sequence can be targeted by engineering a TAL DNA binding domain containing the appropriate order of these modules. The only strict requirement is that each TAL target sequence has a T at its 5’ end.
When expressed in cells, TALEN DNA-binding domains recognize their target sequences within the host genome. If two TALENs are expressed, each binding to a site flanking a 14-20bp spacer, the associated Fokl nuclease domains are able to dimerize and create double-strand break (DSB) within the spacer region between the TAL binding sites. Dimerization of the Fokl domains is required for nuclease activity, so DSB occurs exclusively within the spacer region, and off-target mutations elsewhere in the genome are rare.
Transcription Activator-Like Effector Nuclease (TALEN) architecture: Two TALENs are shown assembled at a target sequence to form a dimeric cleavage complex. Each TALEN consists of an N-terminal TAL DNA-binding domain and a C-terminal Fokl nuclease domain. The TAL domains contain a series of 34-aa repeat modules (shown as colored ovals). Each module is responsible for recognition of a specific DNA base (color-coordinated as indicated at the bottom of the figure). Each target sequence contains a 5’ thymidine, which associates with a cryptic T-binding module (shown in purple) near the N-terminus of the TAL. When dimerized, the Fokl domains introduce a double-strand break (DSB) within the target site.
Cellular repair of DSB by the nonhomologous end-joining pathway (NHEJ) usually results in small deletions, or more rarely insertions and base substitutions. When these mutations disrupt a protein-coding region (e.g. a deletion causing a frameshift), the result is a functional gene knockout. Alternatively, and less efficiently, DSB can be repaired by homologous recombination using exogenous donor DNA template, which is co-introduced with the TALEN vectors. This can result in replacement of the target genomic DNA sequence with template sequence, generating small targeted base changes, such as point mutations, or knockin of large fragments.
Our TALEN knockout vectors contain both a mammalian promoter (e.g. CMV), for TALEN expression by transfection, and a T7 promoter which can be used for RNA preparation by in vitro transcription, for direct injection of RNA into cells or embryos.
For further information about this vector system, please refer to the papers below.
|Nature Protocols7:171 (2012)||TALEN design and function|
|Nat Rev Mol Cell Biol. 14:49-55 (2013)||Review of TALEN technology and potential uses|
|Nat Rev Genet. 15:321-34 (2014)||Review and comparison of TALEN, CRISPR/Cas9, and other genome editing methods|
Our TALEN knockout vectors are designed for quickly and efficiently creating mutations at target sites in a genome. To introduce mutations near a specific target site, a pair of TALEN knockout vectors should be designed with each TALEN binding site flanking the target cut site, separated from one another by 14-20bp, and oriented such that the two TALEN molecules are facing one another while bound to the DNA.
Transient expression: Transfection of the TALEN plasmid vector results in strong transient expression of the TALEN protein within target cells. Without drug selection, the plasmid will be lost over time eliminating the TALEN from the target cells after TALEN-mediated genome editing has taken place.
RNA preparation: In addition to a mammalian promoter, our TALEN knockout vectors also contain a T7 promoter which can be used for RNA preparation by in vitro transcription.
Specificity: TALEN pairs bind opposite sides of the target cut site, and the Fokl nuclease domain requires dimerization for activity,so cleavage at other sites in the genome is unlikely. This high specificity is an advantage over CRISPR/Cas9, which is a less specific genome editing technology.
Two vectors per target site: The Fokl nuclease domain requires dimerization for its function, so a pair of TALEN vectors is required for each target site.
CMV promoter: This drives expression of the downstream TALEN.
T7 promoter: Allow in vitro transcription for RNA preparation.
Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.
Initiation ATG: Start codon of the open reading frame.
3xFLAG: Allows detection of the TALEN fusion protein by immunoassays.
SV40 NLS: Directs the TALEN protein to the cell nucleus.
TAL: Encodes the DNA-binding domain of the TALEN proteins. Consists of the N- and C-termini of the Hax3 TAL and a portion engineered to bind the user-supplied sequence.
FokI: The Fokl nuclease domain from Flavobacterium okeanokoites. It requires dimerization to cleave DNA.
BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.
SV40 promoter: Simian virus 40 enhancer/early promoter. It drives the ubiquitous expression of the downstream marker gene.
Hygro: The Hygromycin resistance gene. This allows cells transduced with the vector to be selected.
SV40 early pA: Simian virus 40 early polyadenylation signal. It facilitates transcriptional termination of the marker gene.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.