Development of a Validated Base-Editor Guide RNA Library for Knockout of Immune-Related Proteins.
Specific protein knockout of immune cells, accomplished by gene editing, can be used to show how certain genes and proteins affect cell function and immune responses. Base editors are Cas9-based enzymes that catalyze single nucleotide genetic changes. Base editors are an ideal alternative to CRISPR-Cas9 nuclease for protein knockout because they are safer and more precise. Here, we tested base editor guide RNA for protein knockout of different targets. To do this, guides for Granzymes A, B, and M, IL10Ra, Granulysin, and Perforin were designed using SpliceR, a web-based program developed by the Moriarity Lab. K562 cells and human primary T and NK cells were electroporated with the guides, then allowed to recover and multiply before collection for downstream assays. Sanger sequencing was used to determine editing efficiency, and flow cytometry was used to determine protein knockout. The results show that a guide for Granzyme A, Granulysin, and IL10Ra achieved >90% editing. The tested guides for other targets did not achieve 90% editing, indicating that there may be factors other than those in the SpliceR prediction software playing a role.