A Technique for Gene Editing in Natural Killer Cells Using CRISPR Cas9

Published: May 31, 2024

Abstract

Source: Naeimi Kararoudi, M., et al. Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins. J. Vis. Exp. (2018).

This video demonstrates a technique for Cas9 ribonucleoprotein-mediated genetic modification of primary natural killer (NK) cells. A Cas9 ribonucleoprotein, consisting of a Cas9 endonuclease bound to a guide RNA (gRNA) formed by base pairing a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), is introduced into primary natural killer cells via electroporation. The ribonucleoprotein targets and cleaves the host DNA at the target site, leading to gene knockout via modification of the target gene through cellular repair machinery.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Human NK Cell Purification and Expansion Isolate peripheral blood mononuclear cells (PBMCs) from Buffy Coat. Layer 35 mL of buffy coat sample on 15 mL of Ficoll-Paque. Centrifuge at 400 x g for 20 minutes without brake and collect the PBMC from the interface. Wash the recovered PBMCs three times by adding at leas…

開示

The authors have nothing to disclose.

Materials

RosetteSep™ Human NK Cell Enrichment Cocktail STEMCELL Technologies 15065 The RosetteSep™ Human NK Cell Enrichment Cocktail is designed to isolate NK cells from whole blood by negative selection.
Ficoll-Paque® PLUS GE Healthcare – Life Sciences 17-1440-02
Alt-R® CRISPR-Cas9 tracrRNA Integrated DNA Technologies 1072532 TracrRNA that contains proprietary chemical modifications conferring increased nuclease resistance. Hybridizes to crRNA to activate the Cas9 enzyme
Alt-R® CRISPR-Cas9 crRNA Integrated DNA Technologies Synthetically produced as Alt-R® CRISPR-Cas9 crRNA, based on the sequence of designed gRNA targeting the gene of intrest
Alt-R® Genome Editing Detection Kit Integrated DNA Technologies 1075931 Each kit contains T7EI endonuclease, T7EI reaction buffer, and T7EI assay controls.
Platinum® Taq DNA Polymerase High Fidelity Invitrogen 11304-011
4D-Nucleofector™ System Lonza AAF-1002B
Human recombinant IL-2 Protein Novartis 65483-0116-07
P3 Primary Cell 4D-Nucleofector™ X Kit Lonza V4XP-3032 Contains pmaxGFP™ Vector, Nucleofector™ Solution, Supplement, 16-well Nucleocuvette™ Strips
Non-targeting: Custom siRNA, Standard 0.05 2mol ON-TARGETplus Dharmacon CTM-360019
Alt-R® S.p. Cas9 Nuclease 3NLS Integrated DNA Technologies 1074181 Cas9 Nuclease
DNeasy® Blood & Tissue Handbook Qiagen 69504
RNeasy Mini Kit Qiagen 74104
Calcein AM ThermoFisher C3099
TGFβ Biolegend 580706
Alt-R® CRISPR-Cas9 Control Kit, Human Integrated DNA Technologies 1072554 Includes tracrRNA, HPRT positive control crRNA, negative control crRNA#1, HPRT Primer Mix, and Nuclease-Free Duplex Buffer.
IDTE pH 7.5 (1X TE Solution) Integrated DNA Technologies 11-01-02-02
Alt-R® Cas9 Electroporation Enhancer Integrated DNA Technologies 1075915 Cas9 Electroporation Enhancer

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記事を引用
A Technique for Gene Editing in Natural Killer Cells Using CRISPR Cas9. J. Vis. Exp. (Pending Publication), e22252, doi: (2024).

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