On the Origins of Homology Directed Repair in Mammalian Cells

Abstract

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.

Keywords: CRISPR; CRISPR-Cas; gene editing; homology directed repair.

Figure 1

The D-Loop. A three stranded reaction intermediate is created when a single-stranded DNA molecule serving as the donor strand for homology directed repair interacts with this complementary strand within the helix. This step comprises the first phase of genetic engineering and DNA pairing. The dynamic binding of the single-stranded DNA with its complement creates the structure known as the D-loop. If a single nucleotide base is engineered into the donor strand so that it creates a mismatch with the nucleotide in the helix. Natural cellular DNA repair enzymes should then act to correct the mismatch and convert a mutant base to a normal base. This step comprises the second phase of gene editing known as DNA repairing.

Figure 2

Regulation of gene editing in mammalian cells. In this diagram, traditional pairing and repairing (or resolution) phases of gene editing are expanded to include specific reactions that take place before, during, and after nucleotide exchange. Importantly, a whole series of metabolic events including DNA replication and DNA repair are active.