Next-generation Disease Modeling
CRISPR-Cas9 gene editing and induced pluripotent stem cells (iPSCs) are both Nobel Prize-winning technologies and represent incredible breakthroughs in their own right. When combined, they become valuable tools for many different areas of research, and they have had a significant impact on modern medicine.
Here, we will cover everything you need to know about CRISPR editing in iPSCs. Specifically, we’ll cover the following key topics:
Let’s get started with the basics of induced pluripotent stem cells.
The advent of induced pluripotent stem cells was a critical breakthrough in medical science. It was the result of decades of research attempting to answer the question “Can we reprogram differentiated cells?” Dr. Shinya Yamanaka won the 2012 Nobel Prize in Physiology or Medicine for his work on iPSCs, sharing the award with Dr. John B. Gurdon, who discovered that the specialization of cells could be reversed more than 40 years earlier.
iPSCs are mature, differentiated cells that have been ‘reprogrammed’ back into a state that is similar to embryonic stem cells. Once they are reprogrammed, the cells become pluripotent, meaning that they can develop into potentially any type of cell. They are usually harvested from skin or blood cells.
iPSCs are produced by treating differentiated cells, such as fibroblasts, with Oct4, Klf4, Sox2, and c-Myc pluripotency-related transcription factors. These genes are introduced to the cells via a retroviral vector to reprogram them into an embryonic-like state from which they can differentiate into any cell type.
The Oct4, Klf4, Sox2, and c-Myc genes are often referred to as ‘the magic four’, the OKSM set, or the Yamanaka factors, after Dr. Yamanaka, the original pioneer of iPSC technology.
iPS cells are used in many areas of research, including disease modeling, developmental biology, drug screening and development, and regenerative medicine. These uses have been improved and expedited by the application of CRISPR-Cas9 technology, which allows for simple and precise gene editing of iPSCs.
iPS cells are especially popular in research as an alternative to embryonic stem cells, which have associated ethical concerns. iPS cells, in contrast, are harvested from mature human skin or blood cells and are not burdened by these ethical concerns.
The discovery of CRISPR-Cas9 gene editing in 2012 by Dr. Jennifer Doudna and Dr. Emmanuelle Charpentier earned them the 2020 Nobel Prize in Chemistry. It was almost immediately apparent to those working in the iPSC field that the conjunction of these two groundbreaking technologies could lead to unprecedented gains in medical research and treatment. In this section, we’ll cover several specific applications of CRISPR editing in iPSCs.
A clear application of iPSC CRISPR technology is functional genomics, the study of what genes do. At the time, researchers employed animal models or immortalized human cell lines to answer questions relating to gene function, using CRISPR or other gene editing technologies, to perform knockouts examining the effects of the edit on the phenotype, behavior, or physiology of the model. However, this approach is limited by the translatability of animal models, epigenetic factors, and the fact that many immortalized cell lines were originally isolated from tumors or harbor mutations.
CRISPR-edited iPS cells eliminate many of the confounding factors associated with these studies, allowing researchers to perform precision studies of gene function on any desired cell or tissue. This is because they can be used to generate isogenic cell lines that are genetically identical except for the particular disease-causing mutation of interest. This also makes these cells incredibly useful for generating much-needed precision models of human disease.
A major challenge in the field of cardiovascular disease research, for example, is to isolate and grow human primary cardiomyocytes used for disease modeling. CRISPR-edited iPSCs can be used to generate cardiomyocytes with specific disease-causing mutations, as well as to correct disease-causing mutations, with relative ease compared to previous methods. They were used in this context in a 2018 study of genomic variants of uncertain significance (VUS), employing the cells as an assessment tool to determine whether VUS are either pathogenic or benign.
They can also be used to generate other cell types involved in cardiovascular disease. For example, a 2017 study used CRISPR to knock out the lipase A (LIPA) gene in macrophages generated from iPSCs to determine the effect on cholesterol ester (CE) handling, efflux, and gene expression, all of which are involved in atherosclerotic cardiovascular disease.
CRISPR-edited iPSCs have also been used to generate much-needed models for studying neurological diseases, such as Alzheimer’s disease (AD), Parkinson’s Disease (PD), and Huntington’s Disease (HD), in which many genetic mutations are implicated. AD, for example, is frequently caused by E280A mutations in the Presenilin1 (PSEN1) gene. In 2019, researchers differentiated iPSCs into two identical neural cell lines, then used CRISPR to induce a homozygous PSEN1 mutation in one cell line and a heterozygous PSEN1 mutation in the other, creating two precision models for AD research.
A notable example use of iPSCs is the 2021 Inducible Pluripotent Stem Cell Neurodegeneration Initiative (iNDI). An initiative by the National Institute of Health (NIH), the iNDI project aimed to generate hundreds of disease models for Alzheimer’s Disease and Related Dementias (ADRD), making it the largest iPSC genome engineering study to date.
Another study generated multiple isogenic iPS cell lines and used them as models to study the effects of various genetic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene, which are associated with inherited PD. The study utilized CRISPR-Cas9 to generate the fourteen cell lines, including both homozygous and heterozygous lines for five different LRRK2 pathogenic mutations, two lines carrying hypothesis-testing mutations, and two LRRK2 knockout lines. Studies like these provide valuable resources for neurological disease research.
A key contributor to the low rates of success in drug discovery research is the lack of reliable disease models. Animal and cell line models, for example, do not reflect the full complexity of human diseases and, as such, are often poorly translated into human clinical studies. Human primary cells and tissues are preferred, however, they are difficult to isolate and maintain in culture, as well as being limited by ethical considerations.
iPSCs removed these roadblocks, by allowing any type of cell to be generated for testing in drug discovery studies. CRISPR-editing the cells can induce any type of mutation required in the iPSCs before differentiation, including gene knockouts (KO), knock-ins (KI), knockdowns (CRISPRi), and activation (CRISPRa). Moreover, CRISPR iPSC protocols are readily scalable, which lends them to high-throughput screening studies, where they can be used to identify novel drug candidates, assess candidate toxicity, or examine the effect of a candidate on disease phenotype.
One study performed high-throughput screening of CRISPR iPS cells to identify potential drug candidates for aging-related diseases that are caused by genetic lesions. Dyskeratosis congenita (DC), one of the diseases investigated, is an incurable bone marrow disorder caused by mutations in several genes that affect telomerase activity and inhibit stem cell proliferation. The study described small-molecule PAPD5 inhibitors, which restore telomerase activity in iPSCs obtained from DC patients. In particular, treatment with BCH001, a novel PAPD5 inhibitor, was able to extend telomeres in CRISPR knockout iPSCs in a dose-dependent manner.
Genome-edited iPS cells have enormous potential in the treatment of disease, and we are already seeing progress toward clinical trials in human patients. Pre-clinical research using CRISPR iPS cells includes novel treatments for diseases such as hemophilia A, Duchenne muscular dystrophy (DMD), beta-thalassemia, diabetes mellitus, and mucopolysaccharidosis.
CRISPR-Cas9 in the field of oncology also incorporates the use of iPSCs. One of these is a 2016 paper that demonstrated a novel application of CRISPRi in enhancing iPSC-derived neural stem cells (NSCs), which are used for the delivery of cancer gene therapies. Because the tumor-tropic ability of NSCs in gene therapy required improvement, CRISPRi was used to alter the activity of a microRNA cluster in the NSCs which regulates hypoxia-stimulated cell migration. After the application of CRISPRi, the NSCs displayed significantly higher levels of tumor tropism, both in vitro and in vivo.
Recent efforts in chimeric antigen receptor T cell (CAR-T) cancer immunotherapy research have focused on generating off-the-shelf, universal solutions that can be used to treat any patient. A key aspect of this is to avoid immune rejection of the delivered T cells. A 2021 paper, published in Nature Biomedical Engineering, used CRISPR to genetically engineer hypoimmunogenic T cells derived from iPSCs, in which allogeneic and cytotoxic immune cell activating factors have been deleted. The resulting cells maintained anti-tumor activity but were able to evade immune responses in mouse models of leukemia, a major step forward in creating a universal CAR-T therapy.
Because iPS cells are able to generate every possible type of adult tissue, they can be effectively used in regenerative medicine and organ transplantation. For patients experiencing organ failure, for example, iPSCs could be used to regrow their own organs, with any disease-causing mutations corrected via CRISPR. While clinical trials of organ transplantation using CRISPR iPS cells are not yet underway, this is certainly a very exciting area of research that could have a significant impact on medical science.
A key proof-of-principle paper from 2017 demonstrated that CRISPR-Cas9 can be used to correct pathogenic mutations in patient-derived iPSCs that have been differentiated into hepatocytes. The study examined homozygous familial hypercholesterolemia (HoFH), a condition characterized by elevated cholesterol and premature cardiovascular disease. HoFH is caused by a three base-pair homozygous mutation in the low-density lipoprotein receptor (LDLR) gene and can be resolved by liver transplantation. The authors of the study used CRISPR to correct the LDLR mutation, after which the hepatocytes showed normal function; these cells can potentially be grown into functional livers for transplantation into HoFH patients.
While the impact and applications of iPSCs are manifold, it currently takes long periods and significant expense to generate and grow iPS cell lines for each and every patient that requires regenerative treatment. In this respect, CRISPR is extremely relevant, because it can be used to easily and precisely edit the genome of iPS cell lines that can be used therapeutically in any patient, eliminating the need to harvest cells from each individual and speeding up the process of preparing tailored treatment. CRISPR interference (CRISPRi) can also be used as an alternative strategy to reprogram iPS cells.
iPSC genetic editing was also relevant during the SARS-CoV-2 pandemic, as it was an attractive tool for infectious disease research. Edited iPS cells can be employed to assess the efficacy of treatment options for Covid-19, to suppress or upregulate certain genes involved in viral infection, or to introduce mutations that could protect against infection. It’s likely CRISPR-Cas9 iPS cells will become more widely used in infectious disease research, particularly in the context of predicted future pandemics.
CRISPR iPS cells are also transforming the landscape of both preclinical research and clinical trials. By enabling the creation of precision models of human disease, iPSC gene editing allows researchers to become less reliant on animal models. This helps to accelerate the entire clinical development process, saving valuable time and resources and allowing studies to progress into clinical trials.
Dr. Dina Simkin's research focuses on using human-iPSCs to model epilepsy. By studying neurons derived from patients with genetic mutations linked to epilepsy, her team aims to understand the disease at a cellular level. This approach allows for more personalized insights into the mechanisms behind epilepsy, potentially leading to better treatments. Her work emphasizes the value of stem cell modeling in uncovering the complexities of neurological disorders.
There are a number of resources available on CRISPR-Cas9 iPS cells, including guides, videos, blogs, review papers, and protocols. Here’s our selection of the most relevant iPSC CRISPR resources on the web.
The use of CRISPR technology in iPSCs has revolutionized the field of gene editing, offering precise and efficient ways to modify pluripotent cells for research, therapeutic development, and disease modeling. However, the potential limitations of CRISPR-Cas9 have spurred interest in exploring high-fidelity alternative nucleases, like hfCas12Max and eSpOT-ON (as recombinant protein or mRNA format), that offer improved specificity and reduced off-target activity. As research progresses, these novel nucleases may further enhance the safety and precision of gene editing in iPSCs, opening new avenues for regenerative medicine and genetic therapies.
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