CRISPR technology enables quick and easy modification of any gene in a given cell type. In a typical CRISPR workflow, target cells are transfected with a plasmid containing both the CRISPR editing construct and a fluorescent reporter, enabling easy identification of successful transfections. Edited cells are then isolated for clonal expansion. Single cell isolation has traditionally been done with two approaches, both of which have inherent drawbacks.
Limiting dilution can be extremely inefficient and costly for clonal cell isolation, particularly when transfection efficiency is low. In the event of low transfection rates, it is nearly impossible to isolate clones via limiting dilution. Moreover, single cell isolation efficiency is highly variable with limiting dilutions, resulting in many empty wells or multiple cells per well.
Fluorescence-Activated Cell Sorters (FACS), while faster and higher throughput than limiting dilutions, operate at high system pressure (up to 70 psi) that can easily damage or kill the sorted cells. Induced pluripotent stem (iPS) cells, a commonly used cell type for CRISPR cloning, are particularly sensitive to the high pressure and have low survival rates following isolation. Additionally, FACS is difficult to operate and prone to contamination.
For CRISPR workflows, cells are transfected with a CRISPR construct and fluorescent reporter, then cultured during a recovery period. Following recovery, fluorescently-labeled transfected cells are identified and dispensed with Namocell’s Single Cell Dispenser. After clonal expansion, colonies are analyzed via PCR to identify which clones contain the desired genotype.
- Gentle sorting (<2 psi) preserves cell viability
- Higher clonality
- Better outgrowth
- 96-well plate in 1 min
- 384-well plate in 6 min
- Quick setup in under 3 min
- No calibration and no maintenance
- Compact size fits in tissue culture hood
- Sterile and disposable cell cartridge
Namocell Single Cell Dispensers are highly efficient and consistent at dispensing single cells. With dispensing efficiency (percentage of dispensed wells with single cells) of 80-90% for CHO cells compared to ~30% using manual pipetting.
Clonal growth of mammalian cells sorted using the Namocell Single Cell Dispenser alongside both limiting dilution (LDC) and FACS. Cells sorted with the Namocell showed higher cloning efficiencies than LDC and FACS.
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