The Powerful Combination of iPSC and CRISPR-Cas9 Genome Editing

Author: Luke Harland Edited by: Sandra Ionescu

Cells are extremely complex, making it a real challenge for scientists to pinpoint what exactly goes awry when disease occurs. Each cell is made up of millions of organic chemicals that work in concert with each other and allow the cell to perform specialised tasks. For example, heart muscle cells contract in synchrony with their neighbours, allowing heart tissue to pump blood continuously to all regions of the body. Heart disease occurs when heart muscle cells no longer fulfil their functional roles. At its core, all human diseases relate in some way or another to cellular dysfunction. It is a primary goal of biomedical scientists to dissect the underlying cellular mechanisms that are disrupted when disease strikes. Understanding these cellular mechanisms enables biomedical scientists to devise targeted strategies to help medical doctors tackle disease in the clinic.

Experimental biomedical science has two primary requirements. Firstly, scientists need to be able to study disease in a setting outside the patient’s body. Usually scientists will study other organisms to learn about how human beings function, but ideally they use human cells for their studies. Furthermore the cellular material they use needs to accurately recapitulate specific attributes of the disease. If a scientist wants to study liver disease, for example, then they require a supply of liver cells. It’s no good trying to study liver disease with pancreas or kidney cells. Secondly, the biomedical scientist needs to be able to precisely alter aspects of the cells they are studying to pinpoint important molecules that play key roles in disease. Each cell of a human body has the same DNA or genetic code. Different cell types utilise this same genetic code in different ways to produce different sets of proteins. Eye cells, for example, produce proteins that enable light detection, whilst intestinal cells produce proteins capable of aiding food absorption. Biomedical scientists, therefore, require tools that allow them to manipulate proteins so that they can learn about their roles in disease. In the past decade, the development of two new innovative biotechnologies has shown potential to revolutionise the way scientists study human disease through provision of these two primary requirements of biomedical experimentation.

The first of these two technologies provide an unlimited supply of human tissue that can be grown in a petri dish. At the earliest stages of human development, you are simple in shape and composition. You initially resemble a spherical ball of cells, much like a golf ball stuffed inside a tennis ball. This primitive structure is called a blastocyst and eventually implants in a mother’s womb. Over the course of the following nine months this primitive ball of cells transforms into a baby with a head, heart, toes and everything in-between. The outer tennis ball structure of the blastocyst contains cells that go on to produce supportive tissues for the development of the fetus. The cells of the golf ball, called the inner cell mass, divide, migrate and specialise into all the cell types of the adult human body.

In the latter part of the 20th century scientists made a remarkable discovery. If you remove the cells of the inner cell mass from a human blastocyst (donated to medical research from IVF clinics) and place them in appropriate growth conditions in a petri dish, they can be propagated indefinitely whilst still retaining their remarkable capacity to specialise into all future cell types of the human body. These cells were called embryonic stem cells (ESCs). Since their initial derivation, many groups have defined protocols that enable the conversion of ESCs into different cell types. Embryonic stem cells, however, only exist transiently in the early human embryo and ethical issues related to their derivation quickly resulted in restricted funding to this promising area of research.

In 2006, Prof Shinya Yamanaka’s research group at Kyoto University circumvented the issues related to embryonic stem cell research by demonstrating the conversion of adult cells into cells much like ESCs. Yamanaka’s group accomplished this cellular conversion via artificial expression of a set of four proteins in adult cells. These protein factors were named the Yamanaka Factors and the resulting converted cells were named induced pluripotent stem cells (iPSCs). iPSCs provided medical scientists for the first time the potential to produce any human cell type from any patient in a petri dish for experimentation.   

Alongside the development of iPSC technology, another new technology burst onto the scene; CRISPR-Cas9 genome editing. CRISPR-Cas9 enables scientists to efficiently and robustly manipulate the genetic code within virtually any human cell. The genome editing works by targeting an enzymatic protein, Cas-9, to specific sections of DNA. Once there, the Cas9 enzyme works like a pair of scissors and cuts the DNA. The cell’s inherent DNA repair pathways can then be exploited to alter the genetic code of the cell. This way, scientists can now study virtually every protein expressed in human cells by robust and efficient manipulation of the genetic code. Previous methods of genome editing were laborious and inefficient. CRISPR-Cas9 in contrast is versatile, efficient and very precise.

Both of these technological advances alone are incredible tools for the biomedical scientist. It is their combinatorial use, however, that promises to provide endless insight into the underlying molecular mechanisms of disease in a patient-specific context. Scientists can now manipulate specific sections of DNA that encode proteins believed to play a role in the disease of a particular patient. By performing this manipulation in patient-derived iPSCs, they are then able to coax these cells into the relevant cell type of the disease-affected tissue. For example, scientists are currently deriving heart muscle cells from patients with heart disease and manipulating genes in them to test hypotheses relating to the functional roles of different genes and proteins in heart disease manifestation. In this way, researchers can start to unravel the cellular mechanisms that are disrupted when disease strikes and provide clues for the development of new therapies that target these root causes.

Another exciting utilisation of these new technologies in tandem relates to the treatment of genetic diseases. Our entire blood system is derived from a specialised cell type called the hematopoietic stem cell (HSC). The HSC can divide indefinitely, reproducing itself whilst also producing precursor cells that mature and develop into all the cells of the blood system. The HSC produces platelets, required for clotting, immune cells that fight disease, and red blood cells that carry oxygen to all our tissues. One of the most common genetic disorders, Beta Thalassemia, afflicts red blood cell function. Beta Thallesemia occurs when there is aberrant production of the beta chains of haemoglobin, the protein required for oxygen transportation. In the future, scientists will be able to derive HSCs from iPSCs of patients with B-thallasemia and utilise CRISPR-Cas9 to fix the mutation that causes the inappropriate production levels of the beta chains of haemoglobin. These altered HSCs can then be transplanted into the bone marrow of patients so that they can produce functional red blood cells. Scientists still need to figure out how to derive HSCs from iPSCs but recently two groups have provided methods that get us tantalisingly close. The general principle of fixing of mutated genes in iPSC cells with CRISPR-Cas9 and the following production of replacement tissue with these iPSCs promises to make personalised medicine a reality in the not so distant future.