Genome Engineering Technology

Genome engineering is a rapidly evolving field that enables the facile manipulation of complex genomes. Genome engineering uses custom endonucleases to create targeted DNA double-strand breaks in genomic DNA. The repair of these DNA breaks can lead to loss-of-function mutations or can be harnessed to create targeted, user-defined genomic modifications.

While this technology first surfaced with the creation of zinc finger nucleases (ZFNs) and was advanced by TALENs, it is now largely being driven by the discovery of the CRISPR (clustered, regularly interspaced short palindromic repeats)/Cas9 system that makes up a novel prokaryotic defense system against nucleic acid invaders (e.g. bacteriophages, plasmids). The CRISPR/Cas9 system includes the Cas9 nuclease, which can be programmed to target and cleave specific sequences in the genome through the use of a guide RNA (gRNA) that directs Cas9 to any user-specified position in the genome. The Cas9 protein binds to the site specified by the gRNA and produces a targeted double-strand break in the DNA. This break is then repaired by the cell and can result in deletions and/or insertions resulting in gene inactivation. Alternatively, if a homologous template is also introduced, repair can occur via homologous recombination-directed repair resulting in precise, user-defined gene editing. This allows investigators to produce mutant cells by modifying a single or many nucleotides in a defined fashion. In addition, investigators can rapidly produce genetically modified mice with the aforementioned genome manipulations by directly injecting Cas9 mRNA and gRNAs into a single-cell mouse embryo. The system is species-agnostic; therefore, it can also be used to produce a variety of modified organisms (e.g. zebrafish, Drosophila, rats). Furthermore, the high efficiency of the Cas9/CRISPR system allows for the introduction of modifications in multiple loci at once.

Induced Pluripotent Stem Cell Technology

In 2007, the laboratory of Shinya Yamanaka demonstrated that human fibroblasts could be “reprogrammed” from their differentiated state to that of pluripotent stem cells by the introduction of four transcription factors (Oct4, c-Myc, Sox-2, and Klf4). James Thomson later demonstrated the ability to accomplish this reprogramming through the introduction of Oct4, Sox-2, Lin28, and Nanog. These reprogrammed cells, referred to as “induced pluripotent stem cells” (iPSCs), have the potential to become any cell type within the body and offer several advantages to other types of human pluripotent stem cells. Most notably, iPSCs circumvent the ethical issues associated with the destruction of human embryos to generate human embryonic stem cells (hESCs). Additionally, iPSCs avoid the problems associated with immune rejection because they are derived from patient-specific cells, thus making them more attractive for regenerative medicine. Finally, iPSCs contain the genetic composition of the patients from whom they were derived and afford the opportunity to study diseases of known genetic disorders. Consequently, iPSCs hold great potential for the study and treatment of human disease.