As a pivotal advance in the gene-editing field and timeline, CRISPR continues to be utilized for research on stem cells and human diseases.
Today, I will explain some of the most notable recent findings in the stem cell-CRISPR field.
To start things off, I have also created an infographic that briefly explains what CRISPR-Cas9 is, what stem cells are, and how they both can be used together to analyze human disease through genetics and potentially treat some specific diseases in the future.
You may also find this review paper on the use of gene-editing in stem cells by Dr. Knoepfler and postdoc Michael Chen to be useful.
A refresher on how CRISPR gene-editing works
CRISPR-Cas9, is a genome-editing mechanism known to be faster, cheaper, and more efficient than previous editing technologies like zinc finger nucleases. It was derived from a natural anti-viral defense system found in bacteria and consists of two main parts—a guide RNA and the Cas9 enzyme—that introduce new sequences into a genome.
The targeted genome could be anything from microbes to plants to humans.
Now on to some specific examples of recent CRISPR gene editing research in stem cells.
Engineered “Invisible” Pluripotent Stem Cells
Scientists at the University of California, San Francisco (UCSF) have genetically engineered pluripotent stem cells that are essentially undetected by the immune system and, therefore, can prevent the obstacle of stem cell transplant rejections. Because cell transplants have the possibility of being seen as foreign and harmful by the immune system, there is a risk of triggering a potent immune response that can harm patients. To avoid this complication and tackle the challenges of induced pluripotent stem cell (iPSC) technology, including cost, genomic instability, and uncertainty of clinical use, UCSF researchers considered designing “universal” iPSCs for any patients.
CRISPR-Cas9 was utilized to alter these IPSCs. The researchers modified three genes in iPSCs, including deleting two genes that control major histocompatibility complexes (MHCs). MHCs are proteins on cell surfaces that display peptides for immune cell recognition, and transplanted organs, tissues, and cells can carry the donor’s antigens that differ from the patient’s antigens and, thus, MHCs can play key roles in evoking a strong rejection reaction.
The team also found a surface protein called CD47 that tells macrophages to not eat (or “phagocytose”) the CD47-displaying cells. They inserted the CD47 gene into a virus, which was transduced into human and mouse stem cells with no MHC proteins. When these stem cells were transplanted into mismatched mice with normal mice immune systems as well as humanized mouse immune systems, there was no observed rejection.
As a result of these modifications, differentiated cells made from the gene-edited iPSCs may avoid detection by human recipients’ immune systems after transplantation in potential future clinical trials. However, such transplanted cells that are largely invisible to the patient’s immune system could pose greater risks if something goes wrong. For instance, if transplanted immune-stealthy lung cells made from the gene-edited iPSCs acquire a cancer-causing mutation, they could have a higher risk of going on to form lung cancer since the patient’s immune system wouldn’t be able to detect them. Thus, researchers will have to monitor for such issues during early phase clinical trials.
CRIPSRi-based Genetic Screens for Human iPSC-derived Neurons
The Kampmann Laboratory at UCSF has conducted genetic screens of human iPSC-derived neurons using a CRISPR variant, CRISPRi. CRISPRi, or CRISPR interference, does not lead to the DNA breaks of the original CRISPR-Cas9 system, which can be toxic to iPSCs. Specifically, the nuclease dead Cas9 (dCas9), led by a single-guide RNA, can target points of interest in the genome, and with it researchers can repress transcription of specific genes in stem cells.
The Kampmann lab used CRISPRi from a safe-harbor site (SHS), a locus in the genome where genes can be safely inserted and not disrupt other genes, to decrease neuron growth without cutting their DNA. More specifically, desired genes were repressed at different points in neuronal differentiation. The researchers carried out CRISPRi-based genetic screens, which can reveal the gene of interest, its defects that may contribute to brain diseases, and the corresponding cellular pathways that can later be targeted for therapeutic purposes. Researchers also found that “housekeeping” genes, or the genes required to survive and essential for all types of cells, behave differently in neurons and stem cells.
As mentioned in the video below, CRISPRi-modified and iPSC-derived neurons suggest that the genetics of brain diseases can be studied without the challenges of animal models, which often do not entirely accurately portray the biology of the human brain, or donor tissue, which have short-lived neurons.
CRISPR for HIV prevention
September of 2019, researchers at Professor HongKui Deng’s laboratory at Peking University published a paper on transplanting CRISPR-Cas9-altered stem cells into a patient with HIV. Specifically, CRISPR was used to modify the genes of donor-derived hematopoietic stem and progenitor cells (HSPCs).
The particular gene of interest was CCR5 because CCR5-null blood cells were shown to be immune to entry of HIV. With the CCR5 mutation as the desired change, Deng’s team used CRISPR-Cas9 to genetically modify the CCR5 gene in the HSPCs and transplant these cells into a patient with HIV-1 and acute lymphoblastic leukemia.
However, after the transplantation, the HIV-1 infection of the patient was not fully eliminated. Despite this, there were still promising results from the experiment: the acute lymphoblastic leukemia diminished within 19 months, and the CCR5-modified stem cells survived with a CCR5 mutation efficiency of 5.20 to 8.28%. Additionally, there were no harmful effects from the gene-editing process.
These recent examples of CRISPR-Cas9 use in stem cells have given insights into disease mechanisms and transplantation biology. There are still challenges with the tool, such as low gene-editing efficiency, off-target binding, low rate of homology directed repair (HDR) compared to nonhomologous end joining (NHEJ), and more. However, CRISPR-Cas9 has become a flexible tool and has been applied to study potential therapeutic methods and targets of various degenerative diseases, including HIV, neurodegenerative diseases, bone/musculoskeletal disorders, cardiovascular disorders, diabetes, and cancer. In addition, CRISPR-related technologies continue to evolve and improve.
You can see the previous post by the author, Mina Kim, a review of 4 cell therapy types under study for COVID-19 here. Dr. Knoepfler also contributed to this post mainly by brainstorming and editing.