The CRISPR CAS9 system is one of the most powerful technologies for cell biology invented in recent years. Persomics has successfully optimized the ImagineArray™ technology for CRISPR CAS9 gene editing with reverse transfection.
Persomics is developing arrayed CRISPR CAS9 screening tools in collaboration with several organisations, including large Pharma and one of the best cell biology research institutes in the world. Access to experience and competence has significantly sped up development.
The Persomics platform is based on reverse transfection, the method invented by David Sabatini at the Whitehead Institute, and Persomics has proven that the same method works exceptionally well for gene editing with synthetic gRNA. Without having fully optimized the conditions, we achieve editing of above 75 % of the cells after 96-120 hours, and typically around 40-50 % after 72 hours in engineered HeLa Cas9 cells.
The proof of concept has been done using CAS9 expressing HeLa and an undisclosed breast cancer cell line. Various transfection reagents from leading providers have been used successfully, and synthetic gRNA from multiple leading tools and reagent companies have been tested and compared.
Persomics ImagineArray is an arrayed screening system with a monolayer of cells ideal for imaging - generating phenotype data analyzed visually as well as measured with data analysis. When choosing genes for proof of concept, Persomics aims to generate cell death, but also show visible phenotypes in living cells. Therefore, one gene (KPNB1) expected to generate cell death was chosen next to Persomics’ benchmark gene (RELA) that produces a distinct cellular phenotype easily detected by the human eye and quantified through image analysis software.
Each cellular component is easily identified through coloured labelling. RNA spots are labelled with red dye, nuclei with blue hoechst and the RELA/p65 protein labelled with green fluorescent antibody. Cells on the control spot show an even distribution of cytoplasmic p65 with a single nuclei in the center of cells. RELA spots show the absence of cytoplasmic green staining in cells indicating that RELA has been edited.
Figure 1, below, shows example of KPNB1 spots with only a few cells left after 72 hours, of which several are about to die.
Figure 1. Editing after 72 h is measured by comparing number of cells per KPNB1 spots to control spots and compensating for apoptotic/dead cells.
Figure 2. Example of RELA spots with good editing after 72 h detected by the lack of the RELA/p65 protein with GFB bound antibodies dye. With the RELA gene cut the cells stays alive with significant lower (or no) production of the RELA/p65 protein.
With tools from Persomics, scientists can focus on the biology and assay development rather than pipetting or configuration of robotics. Persomics provides a robust transfection system generating high levels of editing. The printed reverse transfection experiments remove most of the pipetting steps and mitigate possible mistakes in manual well pipetting. Currently Persomics provides CRISPR screening tools on a custom basis.
So far we have focused on CRISPR based tools with printed synthetic gRNA for screening in CAS9 expressing cell lines, and we will soon start to print plasmids. Be sure to subscribe to our updates to stay informed!
Relevant updates for further reading
- Going forward in reverse – Neil Emans writes about the science behind reverse transfection
- RELA silencing using reverse transfection
- gRNA and CRISPR in reverse transfection (coming)