A TALEN Genome-Editing System for Generating Human Stem Cell-Based Disease Models

Summary: Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease—dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease. Highlights: ► A system for efficient and rapid genome editing with TALENs ► Generation of isogenic human cellular models of disease ► Identification of disease-associated phenotypes in multiple human cell types ► Minimal TALEN off-target effects, but significant clone-to-clone sequence variation Introduction: The study of human disease has been facilitated by the ability to identify the gene mutations responsible; at the same time, it has been hampered by the lack of an inexhaustible supply of easily accessible tissues from patients bearing those mutations. Another limitation is that many gene mutations that would be informative for disease biology if they could be studied in isolated cells are incompatible with human life (i.e., embryonic lethal). Classical gene-targeting technology via homologous recombination has proven to be an invaluable tool of experimental biology through its use in mouse embryonic stem cells for generating germline knockout and knockin mice; however, its use in mammalian systems has been limited primarily to studies in mice. In many cases, mice do not faithfully phenocopy human physiology and disease, e.g., cholesterol metabolism, coronary artery disease, and human hepatitis C virus (HCV) infection. The emergence of genome editing with engineered nucleases, as well as human pluripotent stem cell (hPSC) technology and differentiation protocols to obtain a variety of cell and tissue types in vitro, now make it possible to rapidly interrogate the effects of genetic modification in otherwise isogenic human model systems. Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that, due to their modular domain structure, have proven more straightforward to design and construct for performing genome editing than other types of nucleases (Bogdanove and Voytas, 2011). TALENs are typically designed as a pair that binds to genomic sequences flanking a target site and generates a double-strand break, which is repaired by the cell using either homology-directed repair (HDR) or the error-prone process of nonhomologous end-joining (NHEJ) (Christian et al., 2010; Li et al., 2011; Miller et al., 2011; Hockemeyer et al., 2011). NHEJ can be exploited to introduce small insertions or deletions (indels) resulting in frameshift mutations that effectively knock out a protein-coding gene. An exogenously introduced double-stranded DNA or single-stranded DNA oligonucleotide (ssODN) can serve as a repair template for HDR to incorporate an alteration into the genome (Soldner et al., 2011). In principle, TALEN pairs can be generated de novo with standard molecular biology techniques in a matter of days (Cermak et al., 2011; Sanjana et al., 2012). To demonstrate the utility, efficiency, and rapidity of TALEN technology in generating human cellular models with which to derive new biological insights, we created mutations in 15 genes and performed detailed phenotypic analysis of four genes for which novel roles in disease biology have emerged in recent years—APOB, SORT1, AKT2, and PLIN1. Results: Modular Assembly and Use of TALENs for Efficient and Rapid Genome Editing The DNA-binding domain of a TALEN comprises an array of 33- to 35-amino-acid monomers that are “coded” to recognize and bind specific DNA base pairs (bp) in a 1:1 fashion (Moscou and Bogdanove, 2009; Boch et al., 2009). We built upon previously described modular Golden Gate methodologies to allow assembly of multiple DNA fragments in an ordered fashion (Li et al., 2011; Cermak et al., 2011), such that a single ligation of preassembled tetramers and trimers generates TALENs that recognize any 15 bp recognition site in the genome (Figure 1; Figure S1 available online). This assembly method requires only 1–2 days for completion and is not prone to errors that complicate methods that rely on PCR amplification of monomers. Furthermore, we have developed a set of optimized vectors and methods for the delivery of TALENs into mammalian cells and, in particular, hPSCs. In brief, we transfect or electroporate TALEN pairs into cells and then subject them to fluorescence-activated cell sorting (FACS) 48 hr posttransfection based on fluorescent-marker expression. We replate the sorted cells at low density and allow them to recover and grow for 1 week, resulting in the formation of distinct single colonies. Colonies are expanded, genomic DNA purified, and mutations analyzed by PCR, agarose-gel screening, and Sanger sequencing (Figure 1 and Figure S1). The entire process from start to finish can be completed in less than one month.National Institutes of Health (U.S.) (R01-DK097768