New and TALENted genome engineering toolbox

Abstract

Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the postgenomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.

Keywords: TALENs; cardiovascular disease modeling; genome engineering; induced pluripotent stem cells; zebrafish.

Figure 1

Overview of TALEN genome engineering. Left and right TALEN arms are directed to a specific sequence of genomic DNA by the RVD repeats comprising the DNA binding domain. Specificity is achieved by the one-to-one interactions between the RVDs and nucleotides, as well as a 5’T that is recognized by the N-terminus of the TAL protein. The length of the TALEN arms and spacer region contribute to TALEN activity; here is shown a 15-15-15 (left arm-spacer-right arm) design that has been shown to be effective. The C-terminus domain brings the FokI obligate heterodimer (or homodimer) endonuclease in proximity to dimerize. The resulting double-strand break (DSB) is repaired by either error-prone non-homologous end joining (NHEJ) or programmed gene repair by homologous recombination (HR) or homology-directed repair (HDR) facilitated by an exogenous DNA donor. Repair by NHEJ causes unpredictable insertions/deletions (indels) at the cut site, which can lead to protein loss-of-function. Repair by HR requires long, double-stranded DNA donors that can be used to direct large changes in the genome, such as the fluorescent labeling of an endogenous protein by GFP. Conversely, HDR using short, single-stranded oligonucleotides can be used to make small, subtle changes like a single nucleotide polymorphism (SNP). NLS = nuclear localization sequence; RVD = repeat variable di-residue.

Figure 2

Classes of TAL repeat assembly methodologies. The top panel shows a plasmid-based restriction digest and ligation method that moves TAL repeat monomers (in blue, red, yellow and green) in a pre-determined order from one plasmid to a receiver plasmid by one round of restriction digest and ligation, and then into the destination TAL backbone by another round. The second panel from the top shows a PCR-based restriction digest and ligation method that is conceptually similar to a plasmid-based assembly, however a PCR amplicon replaces whole plasmids as the starting material, and PCR amplification can replace bacterial plasmid propagation. The restriction sites necessary for the digest and ligation reactions are built into the primer design. The next panel outlines a ligation-free assembly method, in which T4 DNA polymerase activity in the presence of dNTPs‘chewsback’ free double-stranded DNA ends to make long single-stranded overhangs. These overhangs anneal efficiently, and bacteria ligate together the DNA backbone if a bacteria origin of replication (grey) and antibiotic resistance gene (black) are included in the reaction. This method eliminates the need for in vitro ligation. The bottom panel demonstrates how solid-state technology can facilitate high-throughput TAL repeat assembly. A biotinylated repeat monomer is first tethered to a plate coated with streptavidin beads, and then monomers are sequentially added until the desired repeat length is achieved. The final assembly is cloned into the final TAL scaffold.

Figure 3

Genome engineering in zebrafish: Modeling the physiology of gene-linked cardiovascular disease. 1) Whole-genome sequencing approaches reveal genetic mutations underlying cardiac disease in an individual patient. Identification of zebrafish orthologs enables TALEN-based gene modification in zebrafish to study the patient-specific defect. 2) Error-prone or programmed gene repair mechanisms facilitate the generation of adult zebrafish with different experimental phenotypes. 3) Engineered zebrafish models provide patient-specific insights into the physiological mechanism of disease. 4) Drug screens offer the potential to identify novel therapeutic agents. 5) Pharmaceuticals are then tested and eventually delivered back to the patient to complete the individualized medicine loop.

Figure 4

Genome engineering in induced pluripotent (iPS) cells: Modeling the cell-autonomous nature of gene-linked cardiovascular disease. 1) Fibroblasts are derived from skin biopsy of patient with an inherited cardiovascular disease. Nuclear reprogramming generates induced pluripotent stem cells that are genetically engineered via TALENs. 2) Error-prone or programmed gene repair mechanisms followed by in vitro cardiac differentiation yield patient-specific cardiomyocytes with different experimental phenotypes. 3) Engineered cardiomyocytes provide patient-specific insights into the cell-autonomous mechanism of disease. 4a) Gene-corrected cardiomyocytes offer an autologous cellular therapy and 4b) safety profiling screens enable the identification of potential arrhythmogenic pharmaceutical agents5) Cellular therapies are delivered back to the patient to complete the individualized medicine loop.