POOL GENERATION / EXPANSION
CONFIRMATION OF EDITS
SCALE-UP FOR APPLICATIONS
Identification of the best method for delivering the CRISPR-Cas9 system into the cells of interest is the first step in the gene editing workflow. When considering which transfection method to use, transfer efficiency and subsequent cell viability are important factors. Transfection efficiency optimization, construct design, delivery method assessment, host line selection are some important factors to be considered.
Transfection efficiency allows researchers to approximate CRISPR/Cas9 gene editing events, providing an early indication for successful gene editing experiments. The IXM imager was used to acquire high quality images post-transfection and pre-antibiotic selection with the (A) Transmitted Light channel, with the (B) green fluorescence channel and (C) red fluorescence channel, and (D) GFP+RFP positive cells. Afterwards, a transfection efficiency can be calculated from the cell counts.
Optimizing GFP transfection with the SpectraMax i3 Multi-Mode Detection Platform
Validating Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 Genomic Editing Experiments Using Molecular Devices Instruments
Optimized for our instruments, we have a wide range of assay kits, labware accessories, and consumables that are designed to lighten your workload while expanding your experiment capabilities.
Ready-to-use assay kits, accessories, and consumables
CloneSelect® Imager FL are a high-throughput automated solution for imaging and analyzing mammalian cells. The all new CloneSelect Imager FL features high contrast multichannel fluorescent and white light imaging that allows for accurate single cell detection and proof of monoclonality at day 0.
CloneSelect Imager FL
Our ImageXpress systems offer an end-to-end solution for high-content screening and analysis. Ranging from automated digital microscopy to high-throughput confocal imaging systems with high-performance lasers, water immersion objectives and proprietary spinning disk technology. Image analysis with machine learning will enable researchers to quickly uncover new insights that are provable and repeatable with clear, accurate data.
ImageXpress High-content Imaging Systems
Pool generation and expansion
Creating a custom gene-modified cell line starts with the evaluation of the transfected cell pool to effectively screen the edited from the unedited in using different selection methods like antibiotic based, fluorescent protein reporter based, antibody tagged cell sorting, and others. The successfully transfected/screened cell pool is then expanded for further monoclonal cell line development.
• Cell pool examination
• Antibiotic or FACS enrichment of successfully transfected cells
• Screen with multichannel feature / confluence
• T-cell pool examination
Plate thumbnails of HEK-293 cells in puromycin selection media, post transfection with CRISPR based plasmids for p53 knockout (various conditions). Reporter signal and confluency measurement were tracked using CSI-FL for further expansion.
Accelerating gene edited cell lines with the CloneSelect Imager FL
Enrichment and single-cell isolation
Enrichment for cells of interest occurs after cells have been transfected. In this step, only those cells that carry the desired edits are identified and expanded. Individual cells are then isolated for confirmation of monoclonality required for regulatory approval.
(A) After a cell-containing sample is loaded into cartridge, the cartridge is mounted onto the SCP and a print run can be initiated. During the printing procedure droplets are continuously generated by a piezo-driven actuator in a predictable sequence. Droplets that contain no cells or more than one cell are siphoned away using a vacuum mechanism directly below the nozzle. When single-cell events are identified, a shutter mechanism inhibits the vacuum allowing the single cell-containing droplet to fall into the well. (B) When a single cell event is detected, five images are captured to provide evidence of clonality at the nozzle. Images 1-3 show the presence of a cell prior to droplet ejection, image 4 marks the region containing the single cell, and image 5 shows nozzle after droplet ejection.
A Workflow Combining Single-cell Isolation and Microplate Imaging improves Cloning efficiency with Higher Assurance of Clonality
The CloneSelect Single-Cell Printer Series is a fully automated system that utilizes proprietary microfluidics-based technology and real-time image analysis to sort and deposit single cells into standard microplates—while simultaneously providing assurance of monoclonality through image documentation. It addresses the shortcomings of limiting dilution and flow cytometry-based cell sorting by offering image-based evidence for clonality, fast and efficient single-cell deposition.
CloneSelect Single-Cell Printer f.sight
Monoclonality verification and growth
Documentation of monoclonality (a regulatory metric for therapeutic cell lines) is typically image-based, whereby an image of a single cell is recorded and included in regulatory filings. Many researchers now routinely use imaging systems, such as the CloneSelect Imager, to verify monoclonality at day 0, and monitor cell growth in cell culture media.
High-throughput screening of up to 384 single-cell clones through label-free or fluorescence-based screening is possible.
Confident Assurance of Clonality using Calcein AM with Minimal Effect on Viability
Verification and functional confirmation of edits
It is important to confirm that target cells have been successfully edited prior to moving on to downstream assays. This can be accomplished either through direct detection of edits using genomic methods or through indirect detection using cellular or proteomic methods. Picking an appropriate assay for your system is the key. Downstream assays for verification and functional confirmation could be picked from various conventional/ NGS methods. Conventional : PCR, Sanger, qPCR, western blot, cell – based assays, etc. NGS : High resolution on and off –target assessment, Single cell RNA- seq, ChIP-Seq, etc
The ScanLater Western Blot detection system generated clear protein bands using time-resolved fluorescence scanning of Eu-bound secondary antibodies. The CRISPR-edited cells demonstrated reduced ATG5 protein expression in comparison to non-edited cells. Vinculin protein was clearly visualized as well. Both sample loading volumes are visible and are not saturated – allowing for quantitative analysis.
ATG5 protein expression
Edited cells showed decreased % relative ATG5 protein expression compared to non-edited cells based on ImageJ analysis. Both loading volumes displayed similar results, implying that there was no overloading/underloading of protein samples
Scale up for Applications
Analyze and Make Discoveries – Phenotype investigation can begin once it has been confirmed that the cells are correctly edited. Further evaluation of the system with a drug as part of a cell-based assay may be desired during target or lead discovery and validation.
Cellular functions-based study
Stable cell lines for preliminary studies related to all applications. (Cell painting data could be used – not limited to this)
Study of disease-causing mechanisms
Identification of effective drugs
This is laboratory-scale research pertaining to all functions and applications
Focuses on data collection, measurement, and analysis.
Manufacture of cellular products
Engineered cells for production:
Cells for therapy or cancer targeting
To cure a disease
Patient-derived cells reprogramming
Drug discovery / Personalized drugs
Stem cell-based gene therapy
Major downstream applications of genome editing but not limited to include :