The Science Of CRISPR/Cas9

Emily BurkeBiologics, Business of Biotech, Cancer, Clinical Trials, Deoxyribonucleic Acid (DNA), Drug Development, Drug Targets, Genetics, Genomics, Mechanism of Action, Ribonucleic Acid (RNA)


As CRISPR/Cas9 adds new indications to its resume, legal battles over its IP continue to be waged in the US and Europe.

On the clinical front, CRISPR/Cas9 entered its first human trial at Sichuan University (Chengdu, China) last fall for metastatic lung cancer, and is widely expected to do so in the U.S. by the end of the year. This past March, a team of scientists at Oregon Health and Science University (Portland, OR) announced that they had successfully edited a gene linked to severe heart defects in human embryos.

On the patent front, CRISPR technology and its applications were discovered by two different research teams, one at University of California, Berkeley, and another at the Broad Institute (Cambridge, MA). Both have filed patents on various aspects of the CRISPR/Cas9 system. The Broad Institute had granted an exclusive license to Editas Medicine (Cambridge, MA), while Berkeley had granted licenses to Caribou Biosciences (Berkeley, CA), CRISPR Therapeutics (Basel, Switzerland and Cambridge, MA),  Intellia Therapeutics(Cambridge, MA), and ERS Genomics (Dublin, Ireland). In February, the U.S. Patent Office ruled in favor of the Broad Institute and its licensee, while in March the European Patent Office ruled in favor of U.C. Berkeley patents. The legal battle is certainly far from over.

With all of these new developments making waves in the industry, let’s review the basics.


CRISPR was originally discovered as a key component of the bacterial immune response. Bacteria, like people, are plagued by viral infections, and bacteria have evolved clever ways to attack invading viruses. In the 1980’s, scientists observed an interesting pattern in bacterial genomes: repeating, palindromic sequences, with unique sequences referred to as “spacers” between the repeats. They dubbed these regions a tongue twister of a name, “clustered regularly interspaced short palindromic repeats,” or CRISPR. Scientists also noticed CRISPR sequences were always located near a gene that coded for an enzyme that cut DNA. This enzyme became known as Cas, short for “CRISPR-associated”.

In the mid-2000’s, scientists realized the “spacers” matched DNA sequences of invading viruses — the bacteria were storing away bits of invading viral DNA between its own bacterial CRISPR sequences!  These bits of viral DNA create a “genetic memory” of the virus, enabling the bacteria to fight back if reinfected.

Reinfection triggers the following steps:

  • Viral DNA present in the spacer sequences is copied into viral RNA.
  • The DNA-cutting enzyme Cas is made, and attaches itself to the viral RNA produced from the spacer sequence.
  • This newly minted viral RNA/Cas complex finds its “match” on the invading viral DNA.
  • The Cas enzyme is now positioned to cut up viral DNA, destroying the invading virus.

In 2013, researchers adapted this bacterial defense for use in human cells. Human cells were engineered to contain both specially-designed RNA and Cas genes. When these human cells produce the RNA/Cas complex, the dynamic duo is ferried to its complementary DNA target. Once in position, Cas goes to work cutting the DNA. The particular Cas protein chosen for this work was one discovered in Streptococcus bacteria, Cas9 — hence the moniker CRISPR/Cas9.

The ability to cut human DNA in precise locations is an exciting innovation because of what the cell does next.


Cas9 creates double-stranded breaks (DSB) in the specified DNA sequence. DSBs cut both strands of the double-stranded DNA helix. Think of DNA as a two-lane bridge that, after experiencing an earthquake, has a section break off and fall into the water below.

DSBs activate two repair pathways to fix 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. 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.

By engineering double-stranded breaks to occur at specific locations, scientists activate the NHEJ or HDR cell repair pathways. By activating the NHEJ pathway, scientists can disrupt a disease-associated gene, preventing the production of a protein that causes the disease. By activating the HDR pathway, a short sequence of DNA is delivered with CRISPR/Cas9 to correct the mutated sequence, perhaps allowing a missing protein that causes disease to be made. In both scenarios cures for many different types of diseases may be realized.


A clinical trial for metastatic lung cancer, initiated last fall by Chinese researchers at Sichuan University use CRISPR/Cas9 to disable the PD-1 gene in T-cells.

The PD-1 gene produces the PD-1 protein, which is located on the T-cell’s surface. When the PD-1 protein is activated, the T-cell doesn’t function. When the PD-1 protein is deactivated, the T-cell functions. Aggressive cancers take advantage of this on/off switch turning PD-1 on, effectively shutting down the T-cell. By turning PD-1 off, the T-cells can’t be suppressed—freeing them up to attack cancer cells.


A U.S. clinical trial of CRISPR to disrupt PD-1 in T-cells is expected to begin before the end of 2017. This two-year study is funded by the Parker Institute (San Francisco, CA).

A number of private companies also have plans for CRISPR/Cas9 clinical trials that include both gene disruption and gene correction. The table below summarizes some key players in the genome-editing arena and their approaches to applying CRISPR. In vivo means the therapy will take place inside the human and Ex vivo means the treatment will be performed in cells taken from the body and then injected back into the patient.

As these and other potential treatments move through clinical trials, the world will be watching to see if this revolutionary technology will live up to the hype and change the way we prevent and treat disease.