文章：

克隆动态限制了在肿瘤异种移植CRISPR/Cas9筛选中选择作用的检测

Clonal dynamics limits detection of selection in tumour xenograft CRISPR/Cas9 screens 

原文发布日期：2023-09-08 

英文摘要：

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. 

摘要翻译： 

可移植体内CRISPR/Cas9基因敲除筛选技术（通过体外编辑细胞后接种于小鼠体内形成肿瘤）能够在包含肿瘤微环境多细胞相互作用的癌症模型中评估基因功能。为深入理解该方法成功的关键参数，我们系统研究了细胞系选择、小鼠宿主、肿瘤采集时间点及向导RNA（gRNA）文库规模等因素。研究发现：当使用GeCKOv2全基因组gRNA文库转导HCT116亚系并移植至NSG小鼠时，在早期（14天）而非晚期（38-43天）时间点采集的肿瘤中，gRNA代表性维持较高水平（80-95%）。晚期肿瘤中gRNA代表性下降的同时，gRNA读数计数方差显著增大，且每个样本中均出现少量随机克隆的明显扩增。这种可变克隆动态产生了高水平"噪音"，限制了基于gRNA选择的检测能力。通过从实验数据生成的模拟数据集，我们证明缩小文库规模可显著降低计数方差。基于这些发现，我们提出了一条合理设计具备充足统计功效的体内CRISPR筛选路径，以实现基因功能的成功评估。

原文链接：

Clonal dynamics limits detection of selection in tumour xenograft CRISPR/Cas9 screens