CRISPR: A Primer

CRISPR/Cas9 Genome Editing for iPSCs

                  Stem cell technology is an area of intense research in biotech as a research tool to study development and disease, but also for the potential therapeutic use of stem cells in repairing damaged tissue and curing disease (“Stem cells: a primer”). Pluripotent stem cells have the ability to divide indefinitely and to turn into any cell type in the body. An important discovery was made just 11 years ago – adult differentiated cells could be reverted or “reprogrammed” into pluripotent stem cells. These induced pluripotent stem cells or “iPSCs” are currently being studied in the lab. In order to be able to fully understand these cells and use them to study and treat disease, we need to be able to make specific changes to their genetic code. For example, we might want to fix a mutated gene that causes disease (such as the mutation which causes the blood disorder beta thalassemia) or we might want to knock out a gene that is causing problems (such as the gene for a receptor that allows HIV to enter a white blood cell).

Gene Editing Before Cut/Copy/Paste

                  Since the 1970s, scientists have been developing tools to make changes to genes. These tools have improved over time but were still limited their ability to make specific changes accurately without side effects. Two of the most powerful of these tools are zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). ZFNs and TALENs have a nuclease, which cuts the DNA strand, and a binding site to help guide the nuclease to the desired location on the genome. However, both of these tools have to be custom-made for each location, which is costly and time-consuming.

Self-Defense for Bacteria

The CRISPR/Cas9 system was originally discovered in bacteria, where it acts as a kind of immune system or self-defense. First, a virus infects a bacterium and introduces its DNA. This DNA is chopped up into pieces which are integrated into the CRISPR site in the bacterium’s DNA (Acquisition). If the same virus tries to infect the bacterium (or one of its descendants) again, the CRISPR DNA is transcribed into crRNA (crRNA Biogenesis). This crRNA combines with a second piece of RNA (tracrRNA) and the enzyzme Cas9, which is responsible for cutting the viral DNA. Since the crRNA matches part of the viral DNA, the crRNA/tracrRNA/Cas9 structure can bind to and cut the viral DNA (Interference). This system is not only specific because of the crRNA, but also simple because the Cas9 enzyme can be combined with any sequence of crRNA to match different genes for cutting.

CRISPR/Cas9 Mechanism. Credit: New England BioLabs Inc.

Room for Improvement

                  CRISPR/Cas9 has revolutionized the field of genome editing in only a few short years. However, scientists have already developed several methods for improving this technique. One of these was the combination of the crRNA with the tracrRNA into a single piece of RNA called the single guide RNA (sgRNA). This makes the system even simpler, going from three components (crRNA/tracrRNA/Cas9) to two (sgRNA/Cas9). Another improvement has been the use of “double-nicking” where two sgRNAs are used to bind to either side of a site on the genome. This allows us to cut out an entire gene and replace it with a new one. One last development has been the creation of a Cas9 nuclease which cannot cut DNA, but instead has added portions which can turn gene on or off. This has been used in the lab to help reprogram cells into iPSCs, but also to differentiate iPSCs into nerve cells.

Credit: New England BioLabs Inc.

The Future

The addition of CRISPR/Cas9 to our gene editing toolbox has the potential to greatly advance iPSC research. We already know of many diseases which are caused by one or two genes, and now we are better able to recreate the process of these diseases using genome editing of iPSCs. Developing cures for diseases using this technique is further down the road, but might be closer than expected. The first clinical trial of the CRISPR/Cas9 system in a human patient took place last year in China, and there is a great deal of hope for the future of this method.