March 1, 2014 (Vol. 34, No. 5)
Jian-Ping Yang Ph.D.
Veronica Blackston Scientist II Life Technologies
Swarup Dash Scientist Life Technologies
Jonathan Chestnut Ph.D. Senior Director Thermo Fisher Scientific
TALEs Enable Rapid and Easy Design of Effectors to Control Gene Expression in Cells
The ability to target a functional protein to a user-defined DNA sequence is a long-sought goal with the potential to enable genome engineering and genetic regulation of diverse cell types. Transcription activator–like effectors (TALE) proteins derived from the bacterial pathogen Xanthomonas have emerged as versatile scaffolds for engineering DNA-binding proteins with user-defined specificity and functionality.
TAL proteins consist of highly conserved N-terminal and C-terminal regions that flank a central domain of tandem nearly identical repeats, which are responsible for the specific DNA binding. Each repeat contains 33 or 34 amino acids and differs from each other mainly in amino acids at position 12 and 13, termed repeat variable diresidues (RVDs).
The RVD of each repeat determines the specificity of binding to a specific nucleotide (A, G, C, or T) in the TALE recognition sequence. Some highly used codes are NI for A-binder, NG for T binder, HD for C binder, and NN for G or A binder. This simple modular DNA recognition allows one to design and construct TALE proteins that target nearly any DNA sequence within the genome in various cells and organisms.
So far, TAL effectors have been successfully used to edit endogenous loci in plants, Drosophila, rat, zebrafish, nematodes, mammals, and yeast.
Using a GeneArt proprietary cloning method for large-scale manufacturing, mammalian codon-optimized TALEs can be rapidly assembled as Gateway entry clones. TALEs in Gateway entry clones offer immediate and easy access to a diverse repository of mammalian and other expression systems.
In addition to standard TALE vectors containing functionalities such as nucleases, activators, and repressors, a multiple cloning site vector has been generated that allows user-specified DNA binding to be combined with any desired activity. Examples of such activities could include a wide variety of DNA and chromatin-modifying enzymes or labeling reagents.
Regulation of Gene Expression by TAL Activators and Repressor
To illustrate the general utility of TAL effectors for regulating chromosomal gene expression in mammalian cells, we first established two stable cell lines in which a single copy of a TAL-responsive cassette was integrated into the genome of FLP-In 293 human embryonic kidney cells (HEK293).
As a reporter for gene activation, the DNA binding target sequence for the wild-type Xanthomonas TALE protein AvrBs3 with 18 bp recognition sequence was inserted upstream of an adenovirus minimal E1b promoter driving a GFP reporter gene (Figure 1A). We also made a chimeric activator by replacing the activation domain of AvrBs3 with a VP16 activation domain from herpes simplex virus. Overexpression of wild-type AvrBs3 TAL effectors or TAL effectors fused with a VP16 activation domain enhanced GFP protein expression approximately five to sixfold from an integrated reporter construct containing only a single TALE binding site (Figure 1B).
In contrast, co-expression of a transcriptional activator GAL-VP16, which targets the S. cerevisae GAL4 gene, failed to activate GFP expression suggesting that activation by wild-type TAL or TAL-VP16 was sequence-dependent.
To initially test TALE-mediated transcriptional repression, we inserted an AvrBs3 DNA binding sequence downstream of a full-length cytomegalovirus (CMV) promoter that drives expression of a GFP reporter gene (Figure 1C).
The TAL transcriptional repressor was generated by replacing the endogenous transcription activation domain of AvrBs with the Kruppel-associated box (KRAB) repression domain of zinc-finger proteins. Overexpression of TAL-KRAB in the repression reporter cells significantly decreased the GFP protein expression after 48 h transient transfection, as indicated by the left-shifted cell population compared to the vector-alone transfection (Figure 1D).
As expected, co-expression of a transcription repressor TetR, which targets the bacterial TetO sequence, had no effect on the GFP expression levels, validating that the repression effect by TAL-repressor is sequence-specific. We have further compared the effect of TAL repressor with that of the short interfering RNA (siRNA), a well-established technology for gene knockdown and repression.
As shown in Figure 1E, siRNA against GFP decreased GFP expression by approximately 60%, which is comparable to the effect observed with TAL effectors. Taken together, these data demonstrate that engineered TALEs can specifically modulate transcription of chromosomal genes in human cells.
Modulation of Endogenous Gene Expression by TAL Activators and Repressors
We designed TAL effectors to target the promoter regions of endogenous Sox2 and ERBB2 genes. The engineered TAL proteins were fused to the synthetic transcription activation domain VP64, a tetrameric repeat of VP16’s core activation domain at the C-terminus.
As shown in Figure 2, TAL activators targeting the Sox2 promoter resulted in a two- to threefold increase of Sox2 transcript levels in 293FT cells and a two- to tenfold increase in HeLa cells 72 h post transfection. In contrast, TAL repressors fused with a KRAB domain decreased the mRNA levels to about 60% compared to vector-alone transfection. A similar observation was made for TAL activators and repressors targeting the ERBB2 promoter (Figure 2B and 2C).
The expression levels of ERBB2 protein detected by anti-ERBB2 antibody were in good agreement with the mRNA data. It is worth noting that the effect of TALEs would be more pronounced if the mRNA and protein levels of target genes were measured from transfected cells as compared to a mixed cell population.
Designed TAL activators and repressors can be used as targeted gene regulators and gene-specific transcriptional switches to turn on or turn off a gene in cells, or to generate novel cellular circuits for biotechnology application.
Targeted Mutagenesis by TAL Nucleases
The modularity of the TALE DNA recognition code enables the design of TAL nucleases to target virtually any DNA sequence in the genome. We generated TAL nucleases by fusing the nonspecific endonuclease domain of FokI to the c-terminus of TAL proteins.
As examples, we engineered TAL nuclease target endogenous adeno-associated virus integration site 1 (AAVS1), hypoxanthine phosphoribosyltransferase (HPRT), and Chromosome 13 sites, which are so-called “safe harbor genomic loci” allowing for safe expression of transgenes in mammalian cells.
All of the tested TALENs were active and the genome-editing activity correlated with the incubation time (Figure 3). Further, mRNA transfection allows earlier and more robust activity of TALEN cleavage. The advantages of mRNA transfection over DNA transfection are that the expression of the TAL nuclease is rapid but transient, and no promoter is required for a specific cell type.
The DSB-based genome engineering can be harnessed to generate gene disruption through nonhomologous end-joining repair, or to promote gene targeting through homologous recombination repair by providing an exogenous donor DNA containing a region homologous to the targeted locus as a template. This allows the introduction of specific changes, such as gene deletion, gene disruption, single-nucleotide polymorphisms, or insertion of entire gene expression cassette into a targeted genomic location. This technology should prove indispensable for the analysis of gene function in cells and animals.
TALEs enable rapid and easy design of effectors to control gene expression in a precise and robust manner in cells. Most recently, a CRISPR-Cas system has been engineered to achieve RNA-guided genome engineering in mammalian cells. These advanced technologies significantly expanded our ability to engineer cells in a directed and combinatorial manner for generating novel cell-based models and therapies.
Jian-Ping Yang, Ph.D. ([email protected]), is a staff scientist, Veronica Blackston is a scientist II, Swarup Dash was a scientist, and Jonathan Chesnut is a research fellow in R&D at Life Sciences Solutions at Thermo Fisher Scientific.