Gene editing based on CRISPR-Cas9 system opens up broad possibilities for the study of the genome and its' transfer to phenotype. The changes of gene will be reflected on the expression and translation processes. Therefore, in case of gene editing we really work with three levels of realization of genomic information - genome, transcriptome and proteome - which allows us not only to describe the function of target gene but also to approach understanding of all cell processes' details.
The scheme of CRISPR-Cas9 system is described as delivering to cell the guide RNA-Cas9 complex via plasmid where guide RNA is complementary to target DNA. The DNA target must be adjacent to a short stretch of sequence termed PAM that is met in DNA approximately every 20-30 nucleotides. And Cas9 endonuclease causes a double strand break at the target site. And in the moment of put the native system of DNA reparation we have an opportunity to add something new to target gene.
Our idea is to add a tag to the gene to further extract the corresponding protein. Using tagged protein is desirable to fish out interacting partners, and it gives hints on protein function. Such hints could be followed by focused functional screens to decipher biological role and activity of proteins under study.
We developed an AP-MS protocol modified with gene editing for HA-tag addition as applied to several protein with uncertain function encoded by genes of human Chr18. For this proteins protein-protein interaction data were obtained in HEK293 cell line under different conditions. As result we showed that this approach is promising for real human interactome construction and protein's annotation due to its' "ecological compatibility" for cell and repeatability.
This work was supported with RSF grant #18-74-00144