Highly efficient transfection of CRISPR/Cas9 using small plasmids

Researchers from SciLifeLab have developed a new method to efficiently deliver the CRISPR/Cas9 machinery into hard-to-transfect human cancer cells and primary blood cells. The study was published in Nature Communications Biology.

The CRISPR/Cas9 technology has revolutionized the field of genome engineering and is used to alter genes and their expression or correct disease-causing genetic variations. Nearly all genes in the human genome can be targeted and the methodology is often used to accelerate disease research.

One of the biggest challenges with the CRISPR/Cas9 system is that the vectors encoding for the CRISPR/Cas9 components are always large (9-19kb) which leads to a low transfection rate and poor cell viability. This means that subsequent selection or purification of positive cells is required.

In a recent study, led by SciLifeLab Fellow Claudia Kutter (Karolinska Institutet), researchers overcame this problem by using an unusual approach in which the cells were co-transfected together with small plasmids of a defined size.

The method is simple, non-toxic and non-viral and resulted in an up to 40-fold increase of the transfection efficiency and an up to 6-fold increase in viability in hard-to-transfect human cancer cells and primary blood cells. The effect also lasted for days.

“Given the broad application of the CRISPR technology (ranging from basic research in biomedicine and clinics, therapeutic and industrial biotechnology), we believe that our method will meet the interest of any researcher using CRISPR for genome engineering and may contribute to accelerate research. We also believe that our method is not restricted to any specific cell type and can be applied to all cells.”, Says Claudia Kutter.