Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.
By Xiquan Liang, J. Potter, Shantanu Kumar, Y. Zou, R. Quintanilla, Mahalakshmi Sridharan, J. Carte, Wen Chen, Natasha Roark, S. Ranganathan and 2 more. Journal of Biotechnology, 2015
701 citations recorded by the source corpus.
CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome modulation in hard-to-transfect cells. The reagent preparation and delivery to cells is amenable to high throughput, multiplexed genome-wide cell engineering.
Subfields: Molecular Biology
View Paper: https://www.semanticscholar.org/paper/2cbe6396a346be7d1e41a18e12d4b311c19bdf66
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Citations in the corpus
- High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes (2015, background): “In mouse embryos and mammalian cells, direct delivery of Cas9 complexes assembled in vitro has been reported to yield a higher frequency of edits (Lin et al. 2014; Aida et al. 2015; Liang et al. 2015).”
- CRISPR/Cas9 in Genome Editing and Beyond. (2016, background): “Commercially available nucleotide transfection reagents have also been adapted to deliver Cas9– sgRNA RNP complexes (256, 257).”
- Engineering the Delivery System for CRISPR-based Genome Editing (2018, background): “They may also exhibit different expression time courses [12], which needs to be considered when designing a therapeutic regimen.”
- Delivering CRISPR: a review of the challenges and approaches (2018, background): “In an intriguing study, Liang et al. (2015) compared the transfection efficacy of three lipofectamine formulations and electroporation against eleven cell lines.”