GENE EDITING IN HUMAN PLURIPOTENT STEM CELLS: CHOOSING THE CORRECT PATH

The recent emergence of targeted nucleases has opened up new opportunities for performing genetic modifications with human pluripotent stem cells (hPSCs). These modifications can range from the creation of a routine knock-out to the more challenging single point-mutation. For both the new and established user, deciding on the best approach for the specific modification of interest can be an arduous task, as new and improved technologies are rapidly and continuously being developed. The choices between the reagents and methodologies depend entirely on the end-goal of the experiments and the locus to be modified. Investigators need to decide on the best nuclease to use for each experiment from among Zinc-Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 that would 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 Fok1 nucleases (RFNs, marketed as NextGENTM CRISPR) that reduces the “off-target” mutation rate, providing further options for investigators. Should researchers need to perform a point mutation, 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. In this review, we will provide a general overview of the current technologies, along with methodologies for generating point mutations, while considering both their pros and cons.