CRISPR Editing in Stem Cells

Thanks to various stimuli that prompt their differentiation, stem cells have the ability to become any cell type. This characteristic makes them great for regenerative medicine, which aims to repair damaged cells, tissues, or organs. Combined with CRISPR editing, which can enable genetic modifications at virtually any location, stem cells have the potential to help scientists understand and treat a variety of diseases. “What makes stem cells really special is the possibility to bring them back into the human body for therapies,” explains Adrian Zambrano, Customer Success Manager in Cytena.

Induced pluripotent stem cells (iPSCs) can be used for patient-specific treatment where iPSCs are edited and reintroduced into the patient. Late last year, the FDA approved CASGEVY™, a CRISPR-edited hematopoietic stem cell therapy, to treat sickle cell disease. CRISPR-edited stem cells have also been used to create organoids, surmounting previous issues in editing cells within organoids.

A bird’s eye view of the CRISPR editing process in stem cells may seem simple: isolate stem cells, make CRISPR edits, screen single cells to find one with the correct edits, expand the single cell into a clonal population, and use these edited cells in your application. However, the use of stem cells—and editing them—can be challenging. We spoke with experts from Cytena, Broken String Biosciences, MilliporeSigma, Caszyme, and Revvity to hear about some of the challenges in CRISPR editing stem cells and how the field is tackling them.

Improvements over ZFNs and TALENs

“In comparison to older genome-editing tools such as ZFN and TALENS, CRISPR/Cas9 has simplified the way editing can be achieved from ease of design and construction, to increased targeting specification and efficiency, with lower cytotoxicity to cells,” says Amanda Haupt, Business Unit Manager, Base Editing at Revvity.

Fong Cheng Pan, Senior Scientist, Translational Research Solutions, at MilliporeSigma, adds, “CRISPR’s precision reduces the risk of unintended mutations compared to zinc finger nucleases and TALENs.”

Still, off-target effects remain

While CRISPR may be more efficient than its predecessors, it still has the potential to produce off-target edits. “To address this, researchers are developing more precise editing tools and methods, such as base editing, to better detect and minimize these off-target activities,” says Tomas Urbaitis, Senior Scientist at Caszyme.

Other strategies include improving gRNA design to minimize mismatches with off-target sites and to optimize screening for gRNAs with the best on-target activity with minimal off-target activity. “Usually what researchers do early in discovery is that they will only focus on on-target activity,” says Elisa Arthofer, Associate Director at Broken String Biosciences, which developed a tool called INDUCE-seq^® to map DNA breaks. “It's important to look at off-target activity at the same time. You don't want to go through several years of development with your chosen guides only to then find that your off-target activities negatively impact your program,” she adds.

Ideally, assessing for off-target edits would rely on reference genomes that represent the entire population. As people around the world have different genetic backgrounds, it’s possible for an off-target edit to be missed if it’s not represented by the reference genomes. Computational tools can help address unrepresented genetic variability and SNPs.

Stem cell characteristics make editing difficult

Stem cells have many characteristics that make them particularly challenging to edit. “Stem cells are unique due to their ability to develop into any type of cell in the body. To maintain this versatility, they have strict mechanisms to keep their DNA stable and avoid passing on mutations to their offspring,” says Urbaitis. Haupt adds, “stem cells are highly sensitive to double-strand DNA breaks.”

This means that clones don’t often survive after editing. “Most of the field is actually working on finding the right supplements and the right coating on the plates to try to increase the probability of survival,” says Zambrano. “In the very best case, you can now expect to reach up to 80% survival.” Cytena is working on improving the probability of stem cells surviving single cell isolation methods.

Another challenge is that they’re highly pluripotent. “They have the potential to differentiate almost into any cell type, and this can really complicate the editing process because as you edit and you make potentially unintended changes to the DNA in your cells, these can have far-reaching effects across different cell lineages,” says Arthofer.

Difficulties in CRISPR delivery

Before editing can happen, the CRISPR components must make it into the cells. Stem cells are less amenable to transfection than other cell types, says Pan. “The way scientists deliver these gene-editing components can cause toxicity or trigger immune responses,” explains Arthofer.

“Stem cells contain high content of heterochromatin, or highly compacted chromatin, which will limit accessibility of CRISPR machinery,” says Pan, who recommends steps to open up the heterochromatin to increase editing efficiency. “However, such manipulation if not done properly can also induce uncontrolled spontaneous differentiation,” she adds.

Excitement for the future of CRISPR editing stem cells

Despite these challenges, there’s still much to be excited about for the future of CRISPR in stem cell editing and regenerative medicine.

CRISPR’s flexibility increases its applications in many different diseases and many types of edits. “CRISPR gene editing can target multiple genes simultaneously, which is a significant advantage when studying complex genetic interaction or for therapeutic intervention that require gene modification at different genomic sites,” says Pan.

Researchers are also using CRISPR to “engineer stem cells with immune-evasive properties so that they can develop personalized cell therapies that are less likely to be rejected by the patient’s immune system,” says Monika Paule, Chief Executive Officer at Caszyme. “CRISPR's potential to customize stem cells for individual patients could also reduce the need for long-term immunosuppressive treatments, further improving patient outcomes and quality of life.”

Haupt is excited about the ability for stem cells to generate a “universal” starting material that can be applied to many therapeutics. “The field has come a long way in understanding how to take cells from a healthy donor and ‘cloak’ them from ever being recognized by a patient’s immune system,” she adds.