Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection

TLDR

CRISPR‑Cas9 offers high‑efficiency genome editing for mammalian cell engineering, yet Cas9 and gRNA delivery remain limiting steps. The study aims to develop rapid gRNA synthesis and Cas9 RNP delivery methods for efficient mammalian cell engineering. The authors prepare Cas9 RNPs and deliver them to mammalian cells using liposome‑mediated transfection or electroporation, enabling high‑throughput, multiplexed genome‑wide engineering. Using these methods, the authors achieved indel rates up to 94 % in Jurkat cells and 87 % in iPSCs, obtained 93 % two‑locus and 65 % three‑locus editing in Jurkat, reduced off‑target cleavage with Cas9 protein versus plasmid, and enabled a four‑day workflow that delivers highly efficient genome modulation even in hard‑to‑transfect cells.

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.