Considerations for engineering a 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 this approach in various 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.

Key considerations for engineering a knockout cell line

To precisely edit or knockout a genomic target, there are some key considerations to be taken into account.

• 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 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.

Alternatives to DIY cell lines

Many labs do not have the time or resources to develop their own cell lines. At Revvity, we offer a range of ready-to-go engineered cell lines to choose from. Alternatively, our expert preclinical services team can develop custom cell lines to your exact specifications, drawing on over a decade of experience in cell line engineering.

Find out more about our custom cell line engineering services or contact our team to discuss your needs.