cat no | io1098
CRISPRi-Ready
ioGlutamatergic Neurons
Human iPSC-derived glutamatergic neurons ready for gene repressions and CRISPRi screens
CRISPR interference (CRISPRi)-Ready ioGlutamatergic Neurons are opti‑ox™ deterministically programmed glutamatergic neurons that stably express catalytically inactive Cas9 nuclease (dCas9) fused to a transcriptional repression domain.
The cells arrive ready for guide RNA (gRNA) delivery from day 1 post-thaw, and high levels of dCas9 expression are maintained for at least 21 days. Using our optimised lentivirus gRNA delivery protocol, users can perform gene repressions, pooled or arrayed CRISPR repression screens and start measuring readouts within a few days.
Place your order
Confidently investigate your phenotype of interest across multiple clones with our disease model clone panel. Detailed characterisation data (below) and bulk RNA sequencing data (upon request) help you select specific clones if required.
Benchtop benefits
Effective repression
Optimised protocol for lentivirus guide RNA delivery in iPSC-derived cells, ensuring repression of target genes.
Ready to use
Highly characterised human neurons stably expressing dCas9 fused to a transcriptional repressor, ready for experiments from day 1.
Quick and easy
Generate readouts within days using a simple protocol for cell maturation and guide RNA delivery.
Go from cell seeding to gene repression to readout in days
Ready for gene repressions
Flow cytometry analysis demonstrates gene repression of CD81 upon lentiviral guide RNA delivery
Flow cytometry analysis confirms robust CD81 gene repression in CRISPRi-Ready ioGlutamatergic Neurons following lentiviral delivery of a CD81-targeting gRNA on day 3 post-thaw. Gene repression was measured by flow cytometry after five days of culture.
(A) 45.8% of cells received the CD81-targeting gRNA via lentiviral transduction, as indicated by GFP expression.
(B) Functionality of the dCas9-based transcriptional repressor is demonstrated by a 5.7-fold decrease in CD81 protein expression (red histogram) compared to non-targeting gRNA controls (grey histogram), measured by geometric mean fluorescence intensity (GMFI) in the GFP+ population.
Repression levels depend on the chosen target and gRNA design
Flow cytometry analysis of CD63 protein expression in CRISPRi-Ready ioGlutamatergic Neurons 5 days after delivery of two different gRNAs. Guide RNAs were delivered on day 3 post-thaw via lentiviral transduction.
Designing gRNAs for CRISPR interference is complex and requires precise targeting of regulatory regions near the transcription start site; efficacy is significantly affected by factors such as chromatin accessibility and epigenetic modifications.
Guide RNA design tools include CHOPCHOP and CRISPick. Our team is also available to help with gRNA design, contact technical@bit.bio.
No silencing of the transcriptional repression domain
Bulk RNA sequencing analysis was performed on CRISPRi-Ready ioGlutamatergic Neurons (CRISPRi) and CRISPRa-Ready ioGlutamatergic Neurons (CRISPRa) at the iPSC stage and on days 1, 7, 14 and 21 post-revival.
Gene expression profiling revealed sustained transcriptional repressor expression in CRISPRi-Ready ioGlutamatergic Neurons throughout the culture period, while no expression of the transcriptional repressor was detected in the control line (CRISPRa-Ready ioGlutamatergic Neurons).
Deliver gRNA from day 1 post-revival
CRISPRi-Ready ioGlutamatergic Neurons are delivered in a cryopreserved format and are programmed to mature rapidly upon revival in the recommended media. The protocol for culturing these cells has two phases: 1. Stabilisation for 4 days 2. Maintenance during which the neurons mature. Delivery of guide RNAs is recommended between day 1 and 11 post-revival. Readout is recommended from 5 days post guide delivery.
Highly characterised and defined
CRISPRi-Ready ioGlutamatergic Neurons form structural neuronal networks by day 11
CRISPRi-Ready ioGlutamatergic Neurons mature rapidly, show glutamatergic neuron morphology and form structural neuronal networks over 11 days, highly similar to wild-type ioGlutamatergic Neurons. Day 1 to 11 post-thaw; 10X magnification.
CRISPRi-Ready ioGlutamatergic Neurons express neuron-specific markers
Immunofluorescent staining on day 11 post-revival demonstrates similar homogenous expression of pan-neuronal proteins MAP2 and TUBB3 (upper panel) and glutamatergic neuron-specific transporter VGLUT2 (lower panel) in CRISPRi-Ready ioGlutamatergic Neurons compared to wild-type ioGlutamatergic Neurons. 10X magnification.
CRISPRi-Ready ioGlutamatergic Neurons demonstrate gene expression of neuronal-specific and glutamatergic-specific markers following deterministic cell programming
Gene expression analysis at day 11 demonstrates that CRISPRi-Ready ioGlutamatergic Neurons (CRISPRi) and wild-type ioGlutamatergic Neurons (WT) lack the expression of pluripotency markers (OCT4 and NANOG), while robustly expressing pan-neuronal (TUBB3 and SYP) and glutamatergic-specific (VGLUT1 and VGLUT2) markers, as well as the glutamate receptor GRIA4.
Gene expression levels were assessed by RT-qPCR. Data normalised to HMBS; cDNA samples of the parental human iPSC line (iPSC) were included as reference; n=3 replicates.
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Giving you access to endless and reliable human cells
“To do a genome-level CRISPR screen, with all the necessary replicates, requires billions of cells. Reaching that scale with iPSCs has been a significant challenge, so, many people turn to immortalised cell lines. But these cells are quite different from neurons in the human body. The development of CRISPR-Ready ioCells is a huge step forward because 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 doing things.”
Emmanouil Metzakopian
Former Group leader, UK Dementia Research Institute, Cambridge University.
VP R&D, bit.bio.
