cat no | io1090
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.
High knockout efficiency
Optimised protocols for lipid or lentivirus based guide RNA delivery ensure maximal knockout efficiency.
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.
CRISPRko-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. gRNAs may be delivered between day 1 and 11, and readouts performed within a few days.
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 CRISPRko-Ready ioGlutamatergic Neurons, that were either transfected or transduced with a gRNA targeting SOX11. gRNAs were introduced into the cells 1 or 3 days after thawing using lentiviral transduction or 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 methods of gRNA delivery. 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 CRISPRko-Ready ioGlutamatergic Neurons 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 methods of gRNA delivery. A non-targeting gRNA was used as a control.
A pooled knockout screen of neurodegenerative disease-relevant genes in CRISPRko-Ready ioGlutamatergic Neurons shows clustering of aaRS genes in UMAPs
For a pooled knockout screen in CRISPRko-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 (aaRS) 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).
CRISPRko-Ready ioGlutamatergic Neurons form structural neuronal networks by day 11
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CRISPRko-Ready ioGlutamatergic Neurons mature rapidly, show glutamatergic neuron morphology and form structural neuronal networks over 11 days, highly similar to wild-type ioGlutamatergic Neurons (io1001). Day 1 to 11 post-thawing; 100X magnification.
CRISPRko-Ready ioGlutamatergic Neurons express neuron-specific markers
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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 CRISPRko-Ready ioGlutamatergic Neurons compared to ioGlutamatergic Neurons (io1001). 100X magnification (upper panel). 200x magnification (lower panel).
CRISPRko-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 CRISPRko-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 normalised to HMBS; cDNA samples of the parental human iPSC line (iPSC) were included as reference; n=3 replicates.
Whole transcriptome analysis demonstrates equivalent expression profiles between CRISPRko-Ready ioGlutamatergic Neurons and wild-type ioGlutamatergic Neurons
Bulk RNA sequencing analysis was performed on two independent lots of CRISPRko-Ready ioGlutamatergic Neurons (CRISPR) and one lot of wild-type ioGlutamatergic Neurons (WT) at day 0 and day 11 of the protocol. Principal component analysis represents the variance in gene expression between CRISPRko-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.
A pooled knockout screen of neurodegenerative disease-relevant genes in CRISPRko-Ready ioGlutamatergic Neurons shows clustering of aaRS genes in UMAPs
For a pooled knockout screen in CRISPRko-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 (aaRS) 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).
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“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.
Consistent. Defined. Scalable.
