Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

To date, lentiviral-based CRISPR-Cas9 screens have largely been conducted in pooled format. However, numerous assays are not amenable to pooled approaches, and lentiviral screening in arrayed format presents many challenges. We sought to examine synthetic CRISPR reagents in the context of arrayed screening. Experiments were performed using aberrant DNA replication as an assay. Using synthetic CRISPR RNAs targeting the known control gene GMNN in HCT-116 cells stably expressing Cas9, we observed statistically significant phenotype among the majority of transfected cells within 72 hours. Additional studies revealed near complete loss of GMNN protein and editing of GMNN DNA. We next conducted a screen of synthetic CRISPR RNAs directed against 640 ubiquitin-related genes. Screening identified known and novel DNA replication regulators that were also supported by siRNA gene knockdown. Notably, CRISPR screening identified more statistically significant hits than corresponding siRNA screens run in parallel. These results highlight the possibility of using synthetic CRISPR reagents as an arrayed screening tool.

截至目前，基于慢病毒的CRISPR-Cas9筛选大多以混合文库筛选模式开展。然而，诸多实验方法并不适用于混合文库筛选策略，且阵列式慢病毒筛选亦存在诸多挑战。本研究旨在探索合成型CRISPR试剂（synthetic CRISPR reagents）在阵列式筛选中的应用潜力。本研究以异常DNA复制作为检测指标开展实验：在稳定表达Cas9的HCT-116细胞中，使用靶向已知调控基因GMNN的合成型CRISPR RNA进行干预，于72小时内观察到多数转染细胞出现具有统计学显著性的表型改变。后续实验进一步证实，GMNN蛋白几乎完全降解，且其基因组DNA发生了编辑。随后，本研究针对640个泛素相关基因开展了合成型CRISPR RNA筛选。此次筛选不仅鉴定出已知的DNA复制调控因子，还发现了全新的调控靶点，且这些结果均通过小干扰RNA（small interfering RNA，siRNA）基因敲低实验得到了验证。值得注意的是，与平行开展的对应siRNA筛选相比，本CRISPR筛选鉴定出了更多具有统计学显著性的阳性靶点。上述研究结果证实，合成型CRISPR试剂可作为阵列式筛选工具。