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. 2020 May 22;11:1008. doi: 10.3389/fimmu.2020.01008

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Copyright © 2020 Huang, Shih, Lai, Chang and Lin.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Screening of the nucleofection conditions for Cas9 ribonucleoprotein (RNP) delivery. (A) Workflow of NK-92 genome editing by Cas9 RNP and DNA nucleofection using a Lonza 4D Nucleofector system. (B) A list of the reference and newly identified nucleofection conditions. (C) Sixteen pulse codes in combination with two buffers, Sol2 and P3, were tested to deliver Cas9 RNP to knockout (KO) CD96 gene. Conditions 1–5 are highlighted in red to show their relative CD96 KO efficiencies and cell viability. (D) Conditions 1–4 were re-examined by a more accurate viability assay. Recovery of viable cells was determined by Precision beads assay and normalized to untreated cells (NT). CD96⁻ cells in the viable population were quantitated. Data are shown as mean ± SD of three independent experiments. Statistics by two-tailed Welch's unequal variances t-test; ns, not significant and ***p ≤ 0.001. The pulse codes, buffers and raw data are summarized in Table S1.