Designer Genes: An Introduction To Genome-Editing

In Biologics, Biotech Basics, Clinical Trials, Deoxyribonucleic Acid (DNA), Drug Targets, Genetics, Genomics, HIV, Mechanism of Action, Ribonucleic Acid (RNA), The WEEKLY by Emily Burke

Five years ago, altering an individual’s genome would have been labeled unimaginable. Fast forward to today and one of the hottest new developments in biotech is genome-editing—the ability to selectively disable or edit the sequence of specific genes.

In this WEEKLY we will compare and contrast the different genome-editing platforms in development and discover how close we are to a clinical reality.

Got Gaps in Your Genes?

Genome-editing is possible thanks to damage in the DNA sequence and the cell’s subsequent activation of different repair pathways. These DNA breaks are  named double-stranded breaks (DSB) because both strands of the double-stranded DNA helix are broken, similar to a two lane bridge that has a section break off into the water below after experiencing an earthquake.

Repair pathways are charged with fixing the break:

    • Non-Homologous End-Joining (NHEJ) closes the gap between the break by joining the two sections back together—imagine pushing the two sides of the bridge together, leaving the fallen section in the water. An unintended byproduct of NHEJ is the possibility of sequence error, much like the sections of the bridge not lining up properly; even a single base deletion may cause unintended consequences. If the repair occurs in the middle of a gene, the minor error can be enough to disrupt gene function and halt the production of the corresponding protein.
  • Homology Directed Repair (HDR) relies on a highly similar (homologous) DNA segment to repair the break – imagine the missing bridge section built elsewhere and helicoptered in to fill the break.

How does genome-editing start? Double-stranded breaks are engineered to occur at specific locations, activating the intrinsic cell repair pathways: HDR and NHEJ.

Catching The Right Break

The secret to genome-editing lies in creating DSBs in therapeutically useful locations with a manmade enzyme called a zinc finger nuclease (ZFN).

How are ZFNs made? To start, zinc finger proteins (ZFP) are sequence-specific, DNA-binding proteins that activate gene expression. They are engineered to recognize unique sites within the genome. While ZFPs do not have the ability to cut DNA on their own, scientists can fuse a ZFP together with a DNA-cutting enzyme called nuclease—the “N” in ZFN. The marriage of ZFP to nuclease creates ZFN.

How do ZFNs work? ZFNs create the desired sequence-specific, double-stranded break within the genome. The DSB activates the NHEJ repair pathway, resulting in disruption of the gene, if there is a desire to “knock out” that gene.  If a repair template is delivered at the same time as the break, the HDR pathway kicks in.

Zinc Finger Nuclease



The biotech world is abuzz with the promise of a new technology with potentially greater flexibility called clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9).

CRISPR/Cas9 is similar in concept to ZFN genome-editing; however,  with CRISPR/Cas9, the nuclease enzyme Cas9 is escorted to the correct location by guide RNAs—or short sequences of RNA complementary to the target sequence. Since producing a new RNA sequence is simpler than producing an engineered protein, targeting the editing site is much easier and faster using the CRISPR/Cas9 system.

Closing the Gap in Genome Editing

ZFN genome-editing is in Phase II trials for treatment of the human immunodeficiency virus (HIV). HIV destroys the immune system by infecting T-cells—a type of white blood cell critical to mounting a defense against invading pathogens. It turns out T-cells function just fine when missing a protein called CCR5, which contains a receptor primarily used by HIV to infect T-cells. Sangamo Therapeutics (Richmond, CA) is using its ZFN genome-editing platform to disrupt the CCR5 gene on patients’ T-cells.

HIV Zinc FInger Nuclease

CRISPR/Cas9 has yet to enter clinical development but several companies, including Editas Medicine (Cambridge, MA) and Caribou Biosciences (Berkely, CA), are in preclinical development of CRISPR/Cas9 technology indicated for a variety of genetic and infectious disease targets.