Discovery on Target (2015) Boston, MA, USA

byX. Liang, J. Potter , S. Kumar, Y. Zou, R. Quintanilla, M. Sridharan, J. Carte, N. Roark, S. Ranganathan, N. Ravinder, and J. Chesnut; Thermo Fisher Scientific, Carlsbad, CA, USA - 09/22/2015

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Introduction

CRISPR-Cas9 systems provide a platform for high efficiency genome editing that can lead to innovative applications in cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remains two steps that limit overall efficiency and general ease of use. Here we describe novel methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA complexes into a variety of cells through liposome-mediated transfection or electroporation, which streamlines cell engineering workflow from gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome editing in hard-to-transfect cells. The reagent preparation and delivery to cells requires no plasmid manipulation so is amenable for high throughput, multiplexed genome-wide cell engineering.

Results

Figure 1. Engineering workflow.
Day 1: design/order CRISPR targets
Day 2: synthesis of gRNA and cell transfection
Day 3-4: genome cleavage assays

Engineering Workflow

Figure 2. gRNA synthesis.
(A) Oligo pool
(B) Synthesis of gRNA template. Lane 1, control. Lanes 2,3 PCR assembly.
(C) In vitro transcription

Figure 3. Lipid-mediated Transfection
(A) Three loci were edited via Cas9 plasmid DNA, mRNA or protein transfection of HEK293FT cells using RNAiMAX. % of Indel was determined. Time course of genome editing (B) and protein expression (C). (D) Off-target mutation of VEGFA T3 target.

Figure 4. Electroporation using Neon™ Transfection System.
(A) Cas9 DNA, mRNA or protein were used to electroporate Jurkat T cells using the Neon 24 optimized protocol, which varies in pulse voltage, pulse width and number of pulses. % of locus-specific cleavage was measured. (B) Dose-dependent effect of genome editing.

Figure 5. Multiplex with cas9 RNPs and 2-3 gRNAs in Jurkat T cells.
Multiplexing assays in Jurkat T cells via cotransfection. Each locus was PCR-amplified from each clonal cell line and sequenced. +/+ indicates % all alleles wt, -/- indicates % all alleles indel, +/- indicates % mixed alleles.

Electroporation using Neon Transfection System

Table 1. Comparison of plasmid DNA, mRNA and Cas9.

Conclusions

  • Design to analysis in 3-4 days using in vitro transcribed gRNA and cas9 protein or mRNA.
  • Cas9 RNPs and cas9 mRNA/gRNAs appear to have lower off-target cleavage rates than plasmid based approaches.
  • Lipid-mediated transfection using Lipofectamine 3000 or RNAiMax works well with some cell lines but more research needs to be done to find proper formulations for difficult cell lines.
  • Electroporation of cas9 RNPs using the Neon Transfection system works in all cells lines tested so far with 20-94% indel efficiency including Jurkat, iPSC, CD34+.
  • Very high indel efficiencies using cas9 RNPs and electroporation enables multiple loci to be targeted simultaneously with minimal downstream screening of clonal isolates (>90% biallelic indels on 2 targets and >60% with 3 targets).
  • The entire process consists completely of liquid handling and enzymatic reaction steps , which make it amenable to higher throughput gRNA production and transfection in multi-well plates.

References

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  4. Kabadi, A.M., Ousterout, D.G., Hilton, I.B. and Gersbach, C.A. (2014) Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Res. Oct 29;42(19):e147. doi: 10.1093/nar/gku749.
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For Research Use Only. Not for use in diagnostic procedures.