Thread by @connor_rosen

Gene modification in primary human NK cells with Cas9 RNP electroporation - very useful preprint @biorxivpreprints - biorxiv.org/content/early/… Highlights and thoughts… #Immunology #GenomeEditing #CRISPR #PreReview #ASAPBio

The bottom line: Best RNP electroporation conditions resulted in up to 80% deletion, consistently >50%, of 3 tested genes for activated primary human NK cells. Validation of Ncr1 and Cish biology from mouse studies in human NK cells.

Paper starts by demonstrating issues with lentiviral transduction in human NK cells. Electroporation was optimized very thoroughly (>10 buffers and pulse conditions), with ~doubling in efficiency (viability and uptake) to >80% transformed and >80% viable

Cas9-GFP plasmids expressed poorly and no deletion was observed. Following trends from T cell literature, they tried Cas9 RNP electroporation. RNP transformation gave >50% homozygous deletion of CD45, stable by flow - no sequencing validation, but flow data are very clear

Now that manipulation of NK cells was established, they validated Ncr1 (coding for NKp46) role in cytotoxicity against Daudi cells (expressing NKp46 binding proteins). Sorted NKp46 -/- cells were less cytotoxic in both donors, matching mouse data

In the process of validating Ncr1, they also demonstrated deletion in expanded (14 days in vitro) NK cells (proliferating, activated, metabolically active) was substantially higher - up to >80% deletion, and ability to titrate efficiency of deletion by sgRNA concentration

For next validation, knockout of Cish. Knockout was robust, and KO cells had enhanced response to IL-15 stim - more proliferation and more JAK1/STAT5 (both total and phosphorylated - consistent with known role of CIS in degrading JAK)

Knockout in primary human NK cells was efficient (NGS quantification of indels was >60% on average, up to 80% varying a bit by donor/prep) and resulted in increased proliferation with IL-15 and increased cytotoxicity

Knockout in cord blood cells (~40% efficiency) and transfer into humanized mice showed normal NK cell differentiation with loss of Cish, and the derived NK cells had enhanced responses to IL-15 and proliferation ex vivo

Overall, extremely thorough optimization and detailed protocols for human NK cell modification, consistent success across 3 loci, validation of targets from mouse biology in human NK cells - great work and thanks to the authors for putting this comprehensive toolkit together!