Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

Area of Science:

• Immunology
• Molecular Biology
• Gene Editing

Background:

• CRISPR/Cas9 gene editing is crucial for engineering cells, but its application in primary natural killer (NK) cells faces challenges.
• Traditional gene delivery methods like viral transduction lead to low yields of genetically modified NK cells due to apoptosis.

Purpose of the Study:

• To establish a DNA-free genome editing method for human primary and expanded NK cells.
• To assess the efficiency of gene knockout and functional modification in NK cells using this novel approach.

Main Methods:

• Utilized Cas9 ribonucleoprotein complexes (Cas9/RNPs) for DNA-free genome editing in NK cells.
• Electroporation was employed for RNP delivery, followed by expansion using feeder cells.
• Targeted gene knockout of TGFBR2 and HPRT1 was performed.

Main Results:

• Achieved efficient knockout of TGFBR2 and HPRT1 genes in NK cells.
• RT-PCR confirmed a significant decrease in targeted gene expression.
• RNP-modified NK cells exhibited reduced sensitivity to TGFβ, as indicated by cytotoxicity assays.

Conclusions:

• The DNA-free Cas9/RNP method provides an efficient means for genome editing in primary NK cells.
• This technique facilitates the generation of functionally enhanced NK cells for potential therapeutic applications.
• Post-electroporation expansion is feasible, supporting the clinical translation of RNP-modified NK cells.