Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells

Authors:
Ayal Hendel, Rasmus O Bak, Joseph T Clark, Andrew B Kennedy, Daniel E Ryan, Subhadeep Roy, Israel Steinfeld, Benjamin D Lunstad, Robert J Kaiser, Alec B Wilkens, Rosa Bacchetta, Anya Tsalenko, Douglas Dellinger, Laurakay Bruhn, Matthew H Porteus
In:
Source: Nat Biotechnol
Publication Date: (2015)
Issue: 10.1038: nbt.3290
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Basic Research
Molecular Biology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
Culture Media:
Platform:
Nucleofector™ I/II/2b
4D-Nucleofector™ X-Unit
Experiment

human T cell: 1 million primary human T cells were nucleofected with 10 µg of the indicated synthetic CCR5 sgRNAs and either 15 µg Cas9 mRNA or 1 µg Cas9-encoding plasmid.

Stimulated T cells: were nucleofected as above, but with 15 µg Cas9 protein complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs

 CD34+ HSPCs from mobilized peripheral blood were maintained in X-VIVO 15 (Lonza) supplemented with SCF (100 ng/ml), TPO (100 ng/ml), Flt3-Ligand (100 ng/ml), IL-6 (100 ng/ml) and StemRegenin1 (0.75 mM).

CD34+ HSPCs were nucleofected using the Lonza 4D-Nucleofector (program EO-100) and the P3 Primary Cell 4D-Nucleofector Kit (V4XP-3024).
Nucleofection conditions: 100 µl nucleofection solution, 5 × 105 cells, 10 µg of the indicated synthetic sgRNAs targeting IL2RG or HBB and either 15 µg Cas9 mRNA or 1 µg Cas9 plasmid. See also 2nd pdf with supplementary information

1 million stimulated T cells or mobilized human peripheral blood CD34+ HSPCs were nucleofected with 15 µg Cas9 mRNA and 10 µg of the indicated synthetic CCR5 sgRNAs. When used in combination the amount of each sgRNA was 5 µg. Before nucleofection, T cells were activated for 3 d with immobilized anti- CD3 antibodies (clone: OKT3, eBioscience, San Diego, CA, USA) and soluble anti-CD28 antibodies (clone: CD28.2, eBioscience).

For non-activated CD3+ T cells, cells were Nucleofected immediately after isolation. T cells were Nucleofected using the Lonza Nucleofector 2b (program U-014) and the Human T Cell Nucleofector Kit (VPA-1002, Lonza).
Nucleofection conditions: 100 µl Nucleofection solution, 106 cells, 10 to 20 µg chemically modified sgRNA,15 to 30 µg Cas9 (or 15 µg eGFP mRNA, TriLink BioTechnologies, San Diego,CA, USA), 1 µg sgRNA/Cas9-encoding plasmid.

Abstract

CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34+ hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.