If you have an electroporator in the lab and reusable cuvettes that would be the easiest method of transfection. If not use calcium phosphate nanoparticles. Ideally you want to use a clonal cell line. Ideally you also want to knock in a selective marker.

When approaching a genome editing goal, it is recommended to begin with the basic HDR strategy, which utilises the canonical CRISPR-Cas9 with either a SpRY or NGG PAM (preferable). There is another category of Cas protein that could amplify the range of accessible PAM, allowing for the creation of a wider range of gRNAs. I do not know if you are familiar with the basics of the CRISPR-Cas system, if not, I hope my remarks are not impolite.

If your goal is to achieve a complete gene knock-out, a two gRNA system will be required. However, if your aim is to suppress gene function, a silencing of the promoter can be accomplished using just one gRNA.

There are numerous methods available, but for a more specific recommendation, further information regarding your case is required.

The use of RNP for CRISPR-Cas delivery is highly recommended, although plasmid delivery is also very efficient and could represent a more cost-effective option.

It is advisable to begin with a pool of cells from which your desired clone can be isolated.

Best of luck!

Thanks for the info, Gaia Gentile Gaia. I had been considering the use of RNP for gene knockout but do you think this is more difficult to achieve compared with plasmid delivery? Also, what method do you recommend for delivery of CRISPR plasmids?

Cheers

Dylan

I used lipofectamine transfection combined with a selectable marker (puromycin or GFP+FACS). My method of choice: (co)transfect two vectors targeting two loci directly adjacent to your GOI, some cells will acquire a full gene deletion. Pick single clones and screen for KO cells using WB or a deletion-specific PCR.