ioGlutamatergic-Neurons-CRISPR-Ready-Hero-Image copy

cat no | ioEA1090 Early Access

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.

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.

per vial

Benchtop benefits

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Ready to use

Defined and characterised human neurons constitutively expressing Cas9, ready for knockout experiments from day 1.

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Quick and easy

Generate readouts within days using a simple protocol for cell maturation and guide RNA delivery.

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High knockout efficiency

Optimised protocols for lipid or lentivirus based guide RNA delivery ensure maximal knockout efficiency.

Go from seeding to knockout to readout in days

 

bitbio-ioCRISPR-webpage_diagrams-V5

 

Technical data

Ready for gene knockouts

Amplicon sequencing demonstrates high knockout efficiency of SOX11 by both lentiviral transduction and lipid-based transfection

ioGlutamatergic-Neurons-CRISPR-Ready-SOX11-KO-Indel-Formation-Amplicon-Seq_solid_bars

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

o	lentiviral transduction of gRNA generates efficient SOX11 knockouts in CRISPR-ready glutamatergic neurons shown by ICC
o	lipofectamine gRNA transfection leads to high-efficiency knockout of SOX11 in CRISPR-ready glutamatergic neurons shown by ICC

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

ioGlutamatergic-Neurons-CRISPR-Ready-Pooled-Screen-scRNA-seq

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

o	Microscopy at 100x magnification shows CRISPR-ready glutamatergic neurons form structural neuronal networks within 11 days

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

o	ICC shows iPSC-derived CRISPR-ready Glutamatergic Neurons express key neuronal markers MAP2, VGLUT2 and TUBB3

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 reprogramming

ioGlutamatergic-Neurons-CRISPR-Ready-RT-qPCR

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

ioGlutamatergic_neurons-CRISPR-Ready-Bulk-RNAseq 3

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.

Cells arrive ready to plate

bit.bio-ioCRISPR_glutamatergic_neurons-timeline-final

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.

Do more with every vial

Industry leading seeding density

ioGlut-MAPT-P301S-P301S-well_plate-1

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. 

Product information

Starting material

Human iPSC line

Karyotype

Normal (46, XY)

Seeding compatibility

6, 12, 24, 48, 96 & 384 well plates

Shipping info

Dry ice

Donor

Caucasian adult male (skin fibroblast)

Vial size

Small: >1 x 10 viable cells

Quality control

Sterility, protein expression (ICC), gene expression (RT-qPCR), Cas9 Protein Activity (ICC)

Differentiation method

opti-ox cellular reprogramming

Recommended seeding density

30,000 cells/cm²

User storage

LN2 or -150°C

Format

Cryopreserved cells

Product use

ioCells are for research use only

Applications

Single gene knockouts
Combinatorial gene knockouts
Pooled CRISPR screens
Arrayed CRISPR screens
High throughput screening

Product resources

Running Large-Scale CRISPR Screens in Human Neurons Webinar
Running Large-Scale CRISPR Screens in Human Neurons

Emmanouil Metzakopian | Vice President, Research and Development | bit.bio

Javier Conde-Vancells | Director Product Management | bit.bio

Watch now
CRISPR-Cas9 knockout screen in iPSC-derived Neurons identifies new Alzheimer’s disease druggable target Publication
CRISPR-Cas9 knockout screen in iPSC-derived Neurons identifies new Alzheimer’s disease druggable target

Pavlou, et al
Nature Scientific Reports
2023

Using CRISPR-Ready ioGlutamatergic Neurons

Read more
CRISPR-Ready ioGlutamatergic Neurons™ User manual
CRISPR-Ready ioGlutamatergic Neurons™
V2
bit.bio
2023
Download
Addressing the Reproducibility Crisis | Driving Genome-Wide Consistency in Cellular Reprogramming Webinar
Addressing the Reproducibility Crisis | Driving Genome-Wide Consistency in Cellular Reprogramming

Dr Ania Wilczynska | Head of Computational Genomics | Non-Clinical | bit.bio

Watch now
Improving Huntington’s disease drug discovery with new reproducible disease models Webinar
Improving Huntington’s disease drug discovery with new reproducible disease models

Dr Emma V Jones | Senior Scientist | Medicines Discovery Catapult

Dr Tony Oosterveen | Senior Scientist | bit.bio

Watch now

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.” 

 

Manos headshot 2Emmanouil Metzakopian
Former Group leader, UK Dementia Research Institute, Cambridge University.
VP R&D, bit.bio.

minus 80 degree freezer for storage-1-1

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