The recent emergence of genome editing technologies has opened up new opportunities for performing genetic modifications in cellular based systems, such as human pluripotent stem cells (hPSCs) as well as animal models, including the rat, a favored model for toxicology. These modifications can range from the creation of a routine knockout to the more challenging knock-ins and single point-mutations. Nucleases that can be employed include Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9), one or a combination could result in the highest likelihood of success with the fewest pitfalls. Furthermore, there have been significant improvements over the first-generation nucleases, such as the development of the dimeric CRISPR RNA-guided nucleases (RFNs, marketed as NextGEN™ CRISPR) that reduces the “off-target” mutation rate, providing further options for gene editing. Should a point mutation or “gene edit” be required, then considerations must be made between using single-stranded oligo deoxynucleotides (ssODN) as the donor for homology-directed repair or utilizing a selection cassette within a donor vector in combination with an excision-only piggyBac™ transposase to leave a seamless edit.

Site-specific nucleases, such as gene editing CRISPR can be used to knockout genes by creating an indel (insertion or deletion) or excise genetic elements, such as enhancers altogether. Another major utility of CRISPR and TALENs in cells and animals is to create a reporter knock-in into a toxicity gene, which permits the use of these cells in toxicity screening experiments. Perhaps the most compelling use for CRISPR, TALENs and excision only piggyBac™ is to create point mutations to model genetic diseases and patient specific cells. This can include creating a mutation that has previously been suggested to correlate with a disease, or to correct a mutation in a patient-derived cell line (1).

Singh et al. (2015) Gene Editing in Human Pluripotent Stem Cells: Choosing the Correct Path. J Stem Cell Regen Biol.