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

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

1. Isolate peripheral blood mononuclear cells (PBMCs) from Buffy Coat.
   1. Layer 35 mL of buffy coat sample on 15 mL of Ficoll-Paque.
   2. Centrifuge at 400 x g for 20 minutes without brake and collect the PBMC from the interface.
   3. Wash the recovered PBMCs three times by adding at least an equal volume of PBS, centrifuging at 400 x g for 5 minutes, and aspirating the PBC wash. NK cell can be isolated at this stage by RosettesSep.
   ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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