On the Origins of Homology Directed Repair in Mammalian Cells

Over the course of the last five years, expectations surrounding our capacity to selectively modify the human genome have never been higher. The reduction to practice site-specific nucleases designed to cleave at a unique site within the DNA is now centerstage in the development of effective molecular therapies. Once viewed as being impossible, this technology now has great potential and, while cellular and molecular barriers persist to clinical implementations, there is little doubt that these barriers will be crossed, and human beings will soon be treated with gene editing tools. The most ambitious of these desires is the correction of genetic mutations resident within the human genome that are responsible for oncogenesis and a wide range of inherited diseases. The process by which gene editing activity could act to reverse these mutations to wild-type and restore normal protein function has been generally categorized as homology directed repair. This is a catch-all basket term that includes the insertion of short fragments of DNA, the replacement of long fragments of DNA, and the surgical exchange of single bases in the correction of point mutations. The foundation of homology directed repair lies in pioneering work that unravel the mystery surrounding genetic exchange using single-stranded DNA oligonucleotides as the sole gene editing agent. Single agent gene editing has provided guidance on how to build combinatorial approaches to human gene editing using the remarkable programmable nuclease complexes known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their closely associated (Cas) nucleases. In this manuscript, we outline the historical pathway that has helped evolve the current molecular toolbox being utilized for the genetic re-engineering of the human genome.