cat no | io1090S
CRISPR-Ready
ioGlutamatergic Neurons
Human iPSC-derived glutamatergic neurons expressing Cas9 for rapid gene knockout generation
CRISPR-Ready ioGlutamatergic Neurons are built from our well-established wild type ioGlutamatergic Neurons, engineered to constitutively express Cas9 nuclease. These cells arrive ready for guide RNA (gRNA) delivery by day 1 post-thaw. Using our optimised lentivirus or lipid-based gRNA delivery protocol, users can maximise their knockout efficiency and start measuring readouts from gene knockouts and CRISPR screens within days.
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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
Ready to use
Defined and characterised human neurons constitutively expressing Cas9, ready for knockout experiments from day 1.
Quick and easy
Generate readouts within days using a simple protocol for cell maturation and guide RNA delivery.
High knockout efficiency
Optimised protocols for lipid or lentivirus based guide RNA delivery ensure maximal knockout efficiency.
Go from cell seeding to gene knockout in days
Ready within days
Schematic overview of the timeline in the user manual
CRISPR-Ready ioGlutamatergic Neurons are delivered in a cryopreserved format and are programmed to rapidly mature 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. gRNAs may be delivered between day 1 and 3 (stabilisation phase). Readouts may be performed within days.
Ready to generate gene knockouts
Amplicon sequencing demonstrates high knockout efficiency of SOX11 by both lentiviral transduction and lipid-based transfection
SOX11 indel formation was measured by amplicon sequencing in CRISPR-Ready ioGlutamatergic Neurons, that were either transfected or transduced with a gRNA targeting SOX11. gRNAs were introduced into the cells either 1 or 3 days after thawing using two methods: lentiviral transduction and synthetic gRNA delivery with Lipofectamine RNAiMAX transfection reagent. After 3 days of culture following guide delivery, DNA was harvested for amplicon sequencing of SOX11. Comparable knockout efficiencies were achieved with both lentiviral transduction and lipid-based transfection. A non-targeting gRNA was used as a control.
Immunofluorescence staining demonstrates high knockout efficiency of SOX11 by both lentiviral transduction and lipid-based transfection
Immunofluorescence staining of CRISPR-Ready ioGlutamatergic Neurons, subjected to a SOX11-targeting gRNA, demonstrates a highly efficient knockout of SOX11. The gRNAs were delivered by lentiviral transduction or transfection of synthetic gRNA using Lipofectamine RNAiMAX on day 1 or day 3 post-revival. Immunofluorescence staining of SOX11 was conducted five days post gRNA delivery. Similar knockout efficiencies were achieved for both lentiviral transduction and lipid-based transfection. A non-targeting gRNA was used as a control.
Neurons at scale for CRISPR screens
A pooled knockout screen of neurodegenerative disease-relevant genes in CRISPR-Ready ioGlutamatergic Neurons shows clustering of aaRS genes in UMAPs
For a pooled knockout screen in CRISPR-Ready ioGlutamatergic Neurons, 100 known genes involved in neurodegenerative diseases were selected. Lentiviral transduction of the gRNAs was carried out on day 3, and single-cell gene expression analysis was performed on day 12. Single cells were clustered on uniform manifold approximations and projections (UMAPs) based on their shared nearest neighbour’s gene expression. Clustering of aminoacyl-tRNA synthetase (aaRSs) knockouts including AARS1, HARS1, CARS1, and GARS1 was observed. In contrast, cells transduced with non-targeting control sgRNAs were evenly distributed among clusters. Pathway analysis showed gRNAs targeting aaRSs activated the unfolded protein response (UPR), the mechanism by which cells control endoplasmic reticulum protein homeostasis. In many neurodegenerative diseases, signs of UPR activation have been reported. The most common aaRS-associated monogenic disorder is the incurable neurodegenerative disease Charcot–Marie–Tooth neuropathy (CMT).
Highly characterised and defined
CRISPR-Ready ioGlutamatergic Neurons form structural neuronal networks by day 11
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CRISPR-Ready ioGlutamatergic Neurons mature rapidly and form structural neuronal networks over 11 days when compared to ioGlutamatergic Neurons (io1001). Day 1 to 11 post-thawing; 100X magnification.
CRISPR-Ready ioGlutamatergic Neurons express neuron-specific markers
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Immunofluorescent staining on post-revival day 11 demonstrates similar homogenous expression of pan-neuronal proteins MAP2 and TUBB3 (upper panel) and glutamatergic neuron-specific transporter VGLUT2 (lower panel) in CRISPR-Ready ioGlutamatergic Neurons compared to ioGlutamatergic Neurons (io1001). 100X magnification (upper panel). 200x magnification (lower panel).
CRISPR-Ready ioGlutamatergic Neurons demonstrate gene expression of neuronal-specific and glutamatergic-specific markers following deterministic programming
Gene expression analysis at day 11 demonstrates that CRISPR-Ready ioGlutamatergic Neurons (CR) and ioGlutamatergic Neurons (WT) lack the expression of pluripotency markers (NANOG and OCT4). In contrast, they robustly express pan-neuronal (TUBB3 and SYP) and glutamatergic-specific (VGLUT1 and VGLUT2) markers, and the glutamate receptor GRIA4. Gene expression levels were assessed by RT-qPCR (data expressed relative to the parental hiPSC control (iPSC Control), normalised to HMBS). Data represents day 11 post-revival samples.
Whole transcriptome analysis demonstrates equivalent expression profiles between CRISPR-Ready ioGlutamatergic Neurons and ioGlutamatergic Neurons
Bulk RNA sequencing analysis was performed on two independent lots of CRISPR-Ready ioGlutamatergic Neurons (CRISPR) and one lot of ioGlutamatergic Neurons (WT) at two different time points (day 0 and day 11) throughout the reprogramming protocol. Principal component analysis represents the variance in gene expression between CRISPR-Ready ioGlutamatergic Neurons and ioGlutamatergic Neurons and shows equivalent expression profiles between these cells. Shapes represent the day the samples from which data was obtained and colours represent the cell type and lot.
Do more with every vial
Industry leading seeding density
CRISPR-Ready ioGlutamatergic Neurons are compatible with plates ranging from 6 to 384 wells.
The recommended seeding density is 30,000 cells/cm2.
This means scientists are able to do more with every vial and expand experimental design within budget without losing out on quality. Resulting in more experimental conditions, more repeats, and more confidence in the data.
One small vial can plate a minimum of 0.7 x 24-well plate, 1 x 96-well plate, or 1.5 x 384-well plate.
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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 ioCRISPR-Ready Cells 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.
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