Ready-to-use iPSC-derived cells transform CRISPR-based functional genomics

CRISPR is a highly effective gene-technology that has revolutionized genomics over the last decade. However, the majority of CRISPR screens rely on immortalized cancer cell lines, which results in models that bear little resemblance to the diseases researchers want to study.

CRISPR-Ready ioCells are the first commercially available iPSC-derived human cells pre-engineered with stable Cas9 expression. These ready-to-use cells eliminate the months-long process of developing CRISPR-compatible cell lines, enabling researchers to perform gene knockout experiments in human neurons, microglia, and motor neurons within days of thawing. This innovation from bit.bio represents a significant shift in the ability to undertake physiologically relevant CRISPR screens, with the potential to drive enormous innovation in drug discovery, target validation and beyond.

Why CRISPR screens started with cancer cells: The scale problem

For years researchers have relied on immortalized cell lines during functional genomics screens. “To do a genome-level CRISPR screen, with all the necessary replicates and controls, requires billions of cells,” explains Dr. Emmanouil Metzakopian, former Chief Scientific Officer at bit.bio and current visiting researcher at the University of Cambridge. “Reaching that scale with iPSC-derived cell types has been a significant challenge, so, many people turn to immortalized cell lines, even though these cells differ substantially from mature, fully functional human cells, such as neurons.”

This has created a gap between the questions researchers want to answer and the suitable models available to them. While immortalized lines like K562, HeLa, and HEK293 offer easy culture conditions and high transfection efficiency, they're particularly poor models for non-oncological diseases, such as neurodegenerative, metabolic, or immunological disorders. These cell lines often lack the cellular context, the desired gene expression patterns, and functional characteristics of the tissues actually affected in human disease, which limits the relevance of the findings.

The alternative, shifting to more biologically representative systems such as iPSC-derived or primary human cells, brings experimental outcomes much closer to human biology but requires months of development. Scientists need to introduce Cas9 into iPSCs, optimize differentiation protocols, and overcome the persistent problem of Cas9 silencing during the transition from pluripotent stem cells to differentiated neurons or other cell types. Even labs with both stem cell and CRISPR expertise often find the process too time-consuming and unreliable for routine use.

bit.bio’s solution was originally developed to support its own screening offering and has quickly developed into a commercially viable product. “Most published screens at the time were done in immortalized cell lines, which are easy to use but lack physiological relevance.” explains Sejla Salic-Hainzl, Vice President of Research and Development at bit.bio discovery. “In the rare cases where iPSC-derived cells were used, CRISPR perturbations were typically introduced at the iPSC stage, before differentiation – with a few key exceptions such as early studies from the Kampmann lab, which demonstrated the potential of functional genomics in iPSC-derived cells.”

Historically, the main barrier to adopting these models has been access: primary cells are hard to source and scale, and generating iPSC-derived models suitable for CRISPR perturbation was technically challenging. “This is what we set out to change with our CRISPR-Ready ioCells, providing scalable, well-defined human cell types with stable Cas9 expression and optimized protocols for gene editing in terminally differentiated cells,” states Salic-Hainzl.

“The development of CRISPR-Ready ioCells is a huge step forward,” adds Dr. Metzakopian. “It allows us to perform large-scale CRISPR screens on cells that closely resemble their in vivo counterparts. It’s a more physiologically relevant way of performing such research.”

Cells as easy as 1, 2, 3

CRISPR-Ready ioCells come cryopreserved in vials, ready to be thawed and cultured according to the easy-to-follow protocol. Optimized protocols are available for both lentiviral guide delivery and synthetic guide RNA transfection, removing another layer of technical optimization. bit.bio ensures that all protocols are optimized so that CRISPR perturbations are performed in terminally differentiated cells, ensuring gene function is studied in a physiologically relevant context. Perturbing at the iPSC stage can mask biology, since essential genes or those affecting pluripotency may prevent cells from ever reaching the differentiated state. In addition, bit.bio’s in-house pooled CRISPR screens consistently show high reproducibility across experiments, underscoring the robustness of the product and CRISPR screening platform.

"We designed CRISPR-Ready ioCells to be as easy and 'plug-and-play' as possible," Salic-Hainzl notes. "Once you receive the cells, thaw them, and plate them according to the user manual, the cells recover quickly and are ready for experiments within a couple of days post-thaw."

These cells are a valuable addition in a wide range of research settings. Neuroscience researchers without CRISPR expertise, for example, can now access gene knockout capabilities without investing months in developing technical competencies. Conversely, CRISPR specialists can upgrade their model systems without learning complex stem cell differentiation protocols.

"Drug discovery teams also benefit, as they can run high-throughput CRISPR screens in human cells like neurons or microglia, improving biological relevance and helping reduce attrition in early-stage development," Salic-Hainzl adds, highlighting the true diversity of these products.

This is a significant advancement for smaller biotech companies and academic labs that lack the resources for extensive cell line development. With the CRISPR-Ready ioCells, months of optimization can now be accomplished in days, with the added reassurance of superior biological relevance.

The concept of perturbing cells in the differentiated state is a well-documented strategy for enabling high-throughput screens that would have been impractical with traditionally derived models^1,2,3.

A growing portfolio built for the future

The current CRISPR-Ready portfolio is broad and continuing to expand, with current CRISPR knockout (CRISPRko)-ready products covering glutamatergic neurons, motor neurons, microglia, and oligodendrocyte-like cells. While CRISPRko-Ready ioCells were bit.bio’s first release, the product portfolio has since expanded to include additional modalities like CRISPRa (activation) and CRISPRi (interference), which allow the target activation or repression of gene expression. This expansion provides researchers with a more complete toolkit for functional genomics.

We are now entering an era where CRISPR-based functional genomics and human cell models are finally converging. This exciting step will see physiological relevant human models being deployed, unlocking insights which were previously inaccessible.

“The ability to run genome-scale CRISPR screens directly in disease-relevant human cells is transformative,” Salic-Hainzl explains. “We’re not just mapping gene functions and interactions, we’re doing it in the right cellular context, and in functional readouts that reflect real disease biology. We’ve already run our first pooled CRISPR screen in a neuron–astrocyte co-culture, and we’re actively working on setting up additional co-culture protocols.”

Looking ahead, comprehensive perturbation datasets across diverse human cell types will be key to powering the next generation of virtual cell models. For researchers ready for the next step CRISPR-Ready ioCells offer a path to more relevant, scalable functional genomics.

“While single-cell atlases have given us detailed snapshots of cell states, what’s still missing is high-quality, large-scale data on how these cells respond to defined genetic perturbations,” concludes Salic-Hainzl. “We hope our CRISPR-Ready cells can contribute meaningfully to this field-by enabling scalable, reproducible, and physiologically relevant functional genomics across a wide range of human cell types.”

References

1. Tian, R., Abarientos, A., Hong, J. et al. (2021) ‘Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis’, Nature Neuroscience, 24, pp. 1020–1034. doi: 10.1038/s41593-021-00862-0.

2. Dräger, N. M., Sattler, S. M., Huang, C. T. L. et al. (2022) ‘A CRISPRi/a platform in human iPSC-derived microglia uncovers regulators of disease states’, Nature Neuroscience, 25, pp. 1149–1162. doi: 10.1038/s41593-022-01131-4.

3. Pavlou, S., Foskolou, S., Patikas, N. et al. (2023) ‘CRISPR-Cas9 genetic screen leads to the discovery of L-Moses, a KAT2B inhibitor that attenuates Tunicamycin-mediated neuronal cell death’, Scientific Reports, 13, p. 3934. doi: 10.1038/s41598-023-31141-6.