Abstract
With the rise of new powerful genome engineering technologies, such as CRISPR/Cas9, cell models can be engineered effectively to accelerate basic and disease research. The most critical step in this procedure is the efficient delivery of foreign nucleic acids into cells by cellular transfection. Since the vectors encoding the components necessary for CRISPR/Cas genome engineering are always large (9–19 kb), they result in low transfection efficiency and cell viability, and thus subsequent selection or purification of positive cells is required. To overcome those obstacles, we here show a non-toxic and non-viral delivery method that increases transfection efficiency (up to 40-fold) and cell viability (up to 6-fold) in a number of hard-to-transfect human cancer cell lines and primary blood cells. At its core, the technique is based on adding exogenous small plasmids of a defined size to the transfection mixture.
Highlights
With the rise of new powerful genome engineering technologies, such as CRISPR/Cas[9], cell models can be engineered effectively to accelerate basic and disease research
We tested if the size of the small vector influences transfection efficiencies by using a range of small vectors (1.8–6.5 kb) (Fig. 1d, Supplementary Fig. 1, Table 2)
The small vector of 3 kb showed the highest increase in transfection efficiency (Fig. 1d, Supplementary Fig. 1f–h)
Summary
With the rise of new powerful genome engineering technologies, such as CRISPR/Cas[9], cell models can be engineered effectively to accelerate basic and disease research. Since the vectors encoding the components necessary for CRISPR/Cas genome engineering are always large (9–19 kb), they result in low transfection efficiency and cell viability, and subsequent selection or purification of positive cells is required. To overcome those obstacles, we here show a non-toxic and non-viral delivery method that increases transfection efficiency (up to 40-fold) and cell viability (up to 6-fold) in a number of hard-to-transfect human cancer cell lines and primary blood cells. CRISPR/Cas has revolutionized genome engineering of biological systems due to its easy design, target site specificity, and scalability for high-throughput applications It allows gene deletions, enhancing or inhibiting gene expression in vitro and in vivo. Due to its easy implementation in current transfection protocols, this strategy may be broadly applicable in basic and applied research
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