title: Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.
abstract: 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.
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Xiquan
authors.initials: X
authors.lastname: Liang
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA. Electronic address: jason.potter@thermofisher.com.
authors.forename: Jason
authors.initials: J
authors.lastname: Potter
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Shantanu
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Zou
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Rene
authors.initials: R
authors.lastname: Quintanilla
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Mahalakshmi
authors.initials: M
authors.lastname: Sridharan
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Carte
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Chen
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Natasha
authors.initials: N
authors.lastname: Roark
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Sridhar
authors.initials: S
authors.lastname: Ranganathan
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Namritha
authors.initials: N
authors.lastname: Ravinder
authors.affiliation: Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Chesnut
mesh_terms: D060846:Cell Engineering / Q000379:methods*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D014162:Transfection
chemical_list: D004720:Endonucleases
keywords: CRISPR; Cas9; Gene editing; Multiplex; Transfection; gRNA