可移植的体内CRISPR/Cas9 敲除筛选是指在 vitro 中编辑细胞，并接种在小鼠体内形成肿瘤，从而评估基因在肿瘤微环境中的功能。为了提高我们对这种方法成功的关键参数的理解，我们研究了细胞系的选择、小鼠宿主、肿瘤采集时间点和引导RNA(gRNA)文库大小的选择。我们发现，当肿瘤在早期(14天)收获时，HCT116亚系转导GeCKOv2全基因组gRNA文库并移植到NSG小鼠体内仍可以保持高gRNA(80-95%)的代表性。但在较晚的时间点(38-43天)，老化肿瘤中的gRNA代表性降低, 同时gRNA读取计数的差异性显着增加，每个样本中随机克隆数目也明显增加。这种变化多样的克隆动力学导致了高水平的“噪音”，限制了基于gRNA的选择的检测。通过使用基于我们实验数据的模拟数据集，我们展示出较小的文库大小可以显著减少计数差异性。根据我们的发现，我们建议一条合理设计足够强大的体内 CRISPR 筛选的途径，以成功评估基因功能。© 2023. 作者。

Transplantable in vivo CRISPR/Cas9 knockout screens, in which cells are edited in vitro and inoculated into mice to form tumours, allow evaluation of gene function in a cancer model that incorporates the multicellular interactions of the tumour microenvironment. To improve our understanding of the key parameters for success with this method, we investigated the choice of cell line, mouse host, tumour harvesting timepoint and guide RNA (gRNA) library size. We found that high gRNA (80-95%) representation was maintained in a HCT116 subline transduced with the GeCKOv2 whole-genome gRNA library and transplanted into NSG mice when tumours were harvested at early (14 d) but not late time points (38-43 d). The decreased representation in older tumours was accompanied by large increases in variance in gRNA read counts, with notable expansion of a small number of random clones in each sample. The variable clonal dynamics resulted in a high level of 'noise' that limited the detection of gRNA-based selection. Using simulated datasets derived from our experimental data, we show that considerable reductions in count variance would be achieved with smaller library sizes. Based on our findings, we suggest a pathway to rationally design adequately powered in vivo CRISPR screens for successful evaluation of gene function.© 2023. The Author(s).