single cell CRISPR analysis in human pluripotent stem cells

Single-cell CRISPR experiment was performed that focused on the pluripotency network in human iPSCs. We selected 23 genes (TFs) that are thought to be involved in pluripotency in hiPSCs and constructed 2 gRNAs per gene. To capture the time-course expression changes, samples were taken from days 2, 3, 4, and 5 after transduction and scRNA-seq analysis was performed. Approximately 5,000 single cells should be captured in each sample to obtain approximately 100 cells per gRNA in the scRNA-seq. Overall design: Human iPSCs (OILG-Cas9) were transduced with the pooled lentivirus for both gRNA and BFP expression at 8-9% transduction efficiency and maintained until harvesting without passaging. On days 2, 3, 4, and 5 after transduction, 8 × 104 BFP+ cells were collected using an MA900 cell sorter (Sony), then resuspended at 1 × 106 cells/mL in 0.05% BSA in PBS. These cells were then subjected to 5 'single-cell RNA-seq library preparation using a Chromium Next GEM Single Cell 5'Library & Gel Bead Kit following the manufacturers' protocol with minor modifications to simultaneously capture guide RNA molecules.

本研究开展了针对人类诱导多能干细胞（human induced pluripotent stem cells, iPSCs）多能性调控网络的单细胞CRISPR筛选实验。我们选取了23个被认为参与人类诱导多能干细胞（hiPSCs）多能性调控的基因（转录因子，transcription factors, TFs），并为每个基因设计构建了2条向导RNA（guide RNA, gRNAs）。为捕捉基因表达的时间动态变化，我们在转导后的第2、3、4、5天采集样本，并进行单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析。为确保在scRNA-seq中每条gRNA对应约100个细胞，每个样本预计捕获约5000个单细胞。
实验整体设计如下：将携带gRNA与蓝色荧光蛋白（blue fluorescent protein, BFP）表达元件的慢病毒混合液转导至人类诱导多能干细胞株OILG-Cas9，转导效率为8%~9%，期间维持细胞培养直至收获，全程未进行传代操作。在转导后的第2、3、4、5天，使用MA900细胞分选仪（索尼，Sony）分选收集8×10⁴个BFP阳性细胞，随后将细胞重悬于含0.05%牛血清白蛋白（bovine serum albumin, BSA）的磷酸盐缓冲液（phosphate buffered saline, PBS）中，调整细胞浓度至1×10⁶ cells/mL。随后使用Chromium Next GEM单细胞5'端RNA测序文库制备试剂盒及凝胶磁珠试剂盒，参照厂商官方实验方案并进行小幅修改，对上述细胞进行单细胞5'端RNA测序文库构建，以实现同时捕获向导RNA分子。