Ready. Set. CRISPR.

In Biotech Basics, Business of Biotech, Cancer, Deoxyribonucleic Acid (DNA), Easily Confused, Genetics, Genomics, HIV, Ribonucleic Acid (RNA), The WEEKLY by Emily Burke

CRISPR/CAS9 TECHNOLOGY BUILDS UP STEAM

The world definitely weighed in after Chinese scientists published a paper detailing the use of CRISPR/Cas9 to edit nonviable human embryo genomes. While the embryos used were never intended to become a living human being, this controversial milestone created a firestorm of opinion.

Tweets, blogs, and mainstream news stories were abuzz about the opening of Pandora’s box. Even Jennifer Doudna, one of the creators of the technology, called for a moratorium. Just today, NIH stated they will not fund the use of embryos in genome-editing research. In the end, CRISPR/Cas9 did not successfully edit the embryo genomes as intended, which underscores the juvenescence of the technology.

The brave new world of genome-editing continues to intrigue us here at WEEKLY. What is it about the science of CRISPR/Cas9 that is creating a surge of investor euphoria? Let’s take a look at the science behind the controversy.

BREAKING AND FIXING A GENE

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. Think of a two lane bridge that, after experiencing an earthquake, has a section break off and fall into the water below.

Two kinds of repair pathways are charged with fixing the break in the DNA:

  • Non-Homologous End-Joining (NHEJ) closes the gap between the break by joining the two sections back together—visualize 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—visualize the missing bridge section built elsewhere and helicoptered in to fill the break.

While these repair pathways are the body’s natural way of fixing a break in the genome, the process has been hacked by scientists. Double-stranded breaks are engineered to occur at specific locations, activating the intrinsic cell repair pathways of NHEJ and HDR.

CRISPR/CAS9 ON THE RUN

The secret to good genome-editing lies in creating DSBs in therapeutically useful locations within the genome. CRISPR/Cas9 technology enables just that.

CRISPR stands for “clustered regularly interspaced short palindromic repeats”. These are brief DNA sequences that read the same forward and backwards (palindromic repeats).

In the mid-2000s, scientists realized palindromic sequences were an integral part of the bacteria’s immune system.  Bacteria store away bits of invading virus DNA between its own CRISPR (DNA) sequences for memory purposes. If a virus eventually infects the bacteria, the previously stored away viral DNA is copied and used to make viral RNA. When the viral RNA is released, it recognizes and binds to the DNA of the subsequent invading virus. The binding action triggers the protein Cas9 to cut up the viral DNA, knocking out the virus.

In 2013, researchers showed they could use this same concept in animal cells. They simply designed sequence-specific RNA to escort Cas9 to a specific DNA site. Once at the site, Cas9 cuts the DNA sequence. This DSB results in disruption of the specific gene and activates either the NHEJ or the HDR repair pathway.

EASILY CONFUSED: ZFN VS. CRISPR

Zinc-finger nuclease (ZFN) is a protein engineered to bind and cut a specific genome sequence. CRISPR technology, on the other hand, relies on a RNA guide sequence to identify and escort another protein (Cas9) to the correct genome sequence. The Cas9 protein is then responsible for the actual cutting of the selected location.

CRISPR intended RNA sequences are quicker and less expensive to produce, as compared to engineering ZFN proteins. However, ZFNs have a longer track record and are currently in the clinic being tested as a potential cure for HIV/AIDS.

THE CRISPR/CAS9 LINE UP

Early, well-financed players in the CRISPR/cas9 arena include Intellia Therapeutics (Cambridge, MA), and CRISPR Therapeutics (Basel, Switzerland).  Both announced plans to focus first on applications that involve modifying cells (like blood or bone marrow) outside of the body. The cells will be reinjected, bypassing (for now) the challenge of delivering the RNA guide sequences and Cas9 protein into cells within the patient’s body. Intellia Therapeutics licensed the technology from Caribou Biosciences (Berkeley, CA), which is also a contender in the CRISPR/Cas9 line up.

Others are working on CRISPR/Cas9 delivery systems. During fall 2014, Editas Medicine (Cambridge, MA) published successful results on the use of adeno-associated viral (AAV) gene therapy vectors to deliver CRISPR/Cas9 to disrupt the expression of three target genes in the neurons of mice. Editas recently licensed a Harvard-developed lipid-based delivery technology for CRISPR/Cas9.

Researchers are also developing CRISPR/Cas9 applications to fight infectious disease: Emory University to combat hepatitis C virus and MIT to treat fungal infections. Other early-stage academic research projects include targeting cystic fibrosis (Yale) and epigenome editing (Duke).

With the race heating up, CRISPR/Cas9 is widely expected to be utilized in a whole range of diseases, including cancer, immune disorders, blood disorders, rare genetic diseases, and even infectious diseases.