Engineering a Knockout Cell Line
Considerations for successful knockout cell line generation
CRISPR-Cas9 has made it easier and faster to create an engineered cell line, giving rise to the popularity of the approach in a number of experimental areas, such as protein-protein interaction, hit validation, genomic functional analysis, and creation of disease models. Applications such as protein tagging also help enrich our understanding of particular gene targets. To precisely edit or knockout a genomic target, there are several considerations to be taken into account. Our Cell line engineering webinar goes into these topics in detail, here is a quick look at what it covers:
- Choice of cell line – What is the most suitable cell line to model a disease or test a gene’s functional effect? In addition to biological origin, you must consider the difficulty of delivery of CRISPR components, whether the cells can be enriched for targeted events, and the tolerance of the cells to the dilution steps necessary to generate a clonal population.
- CRISPR-Cas9 reagent selection – In addition to selection of a highly functional guide RNAs to create a double-strand break within your gene target, you will need to optimize the delivery of Cas9 nuclease to your cells for maximum efficiency. If you’re performing a knock-in (introduction of a SNP, fluorescent tag, or other alteration) the design of an oligo or plasmid donor is critical to experimental success.
- Screening of clones – How amenable are your cells to dilution, and how many clones do you need to analyze to find those that are homozygous for the gene editing event? What method will you use to identify edited cells? This is potentially the most critical aspect of cell line development since the isolation and characterization is when your hard work in the CRISPR experiments will culminate.
- Validation – Initial characterization of edited cells must be followed by more thorough validation, most often using protein-level methods (Western blot, immunofluorescence). The method(s) will be driven by the genomic alteration being made.
Horizon catalog cell lines
If you would like to save the time and effort involved in doing your own cell line engineering, you should review the catalog of ready-made cell lines available from Horizon Discovery. The catalog contains our proprietary HAP1 cell lines, plus an expanding range of edited cell lines that include common cancer cell lines such as DLD1, MCF10a and HCT116.
The available knockout cell lines have complete loss of function for the target gene, and knock-in cell lines can improve understanding of a disease gene’s role in a phenotype from relevant mutations.
Authors: Louise Baskin, Senior Product Manager
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