Genome-wide CRISPR screen for regulators of SARS-CoV-2 programmed ribosomal frameshifting

Genome-wide CRISPR-Cas9 knockout screens were performed in dual-fluorescent frameshifting reporter cell lines to identify human host factors for SARS-CoV-2 programmed ribosomal frameshfiting. Overall design: Two independent clones for PRF-1 and PRF0 were used for the screen and 1.4x10^8 cells were infected with the Brunello pooled lentiviral CRISPR library (Addgene #73179) in the presence of 5 µg/ml polybrene at a multiplicity of infection between 0.3 to 0.5. Two days after transduction, the cells were selected in medium containing 1 µg/ml puromycin. After 10 days of puromycin selection, the top 1% eGFP/mCherry population and bottom 0.75% eGFP/mCherry population were sorted on an FACS Aria 2 cell sorter (BD Biosciences). gDNA from sorted pools was isolated by phenol-chloroform extraction. gDNA from 6x10^7 unsorted cells, was isolated with the Masterpure DNA isolation kit (Lucigen). Sequencing libraries were generated from the isolated gDNA through two sequential PCR reactions using the Herculase II DNA polymerase (Agilent). After 18 rounds of initial PCR amplification, 5% of the reaction product was used for a second round of PCR (11-13 cycles) with primers that introduced Illumina sequencing adapters and barcodes. The final PCR products were purified using AMPure XP beads (Beckman Coulter). Libraries were sequenced on a NextSeq500 (Illumina) with 75 bp single-end reads at an average sequencing depth of ~2x10^7 reads per sample. sgRNAs sequences were extracted from fastq files through an in-house Galaxy script and normalized reads counts were calculated.

本研究采用双荧光移码报告细胞系（dual-fluorescent frameshifting reporter cell lines）开展全基因组CRISPR-Cas9敲除筛选（Genome-wide CRISPR-Cas9 knockout screens），以鉴定严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）编程性核糖体移码（programmed ribosomal frameshifting）的人类宿主因子。
实验设计概述：本筛选使用PRF-1与PRF0的两个独立克隆，取1.4×10^8个细胞，在5 μg/ml聚凝胺（polybrene）存在下，以感染复数0.3~0.5感染Brunello pooled lentiviral CRISPR文库（Addgene #73179）。转导两天后，使用含1 μg/ml嘌呤霉素（puromycin）的培养基对细胞进行筛选。嘌呤霉素筛选10天后，通过FACS Aria 2细胞分选仪（BD Biosciences）分选得到eGFP/mCherry信号最高的1%细胞群与最低的0.75%细胞群。
分选得到的细胞群的基因组DNA（gDNA）采用酚-氯仿抽提法提取；另取6×10^7个未分选细胞，使用Masterpure DNA提取试剂盒（Lucigen）提取基因组DNA。通过分离得到的gDNA，利用Herculase II DNA聚合酶（Agilent）经两轮连续PCR反应构建测序文库：首轮PCR扩增18个循环后，取5%的反应产物作为模板进行第二轮PCR（11~13个循环），所用引物可引入Illumina测序接头与条形码序列。最终PCR产物采用AMPure XP磁珠（Beckman Coulter）纯化。测序文库在NextSeq500测序仪（Illumina）上进行75 bp单端测序，每个样本的平均测序深度约为2×10^7条读段。通过自研Galaxy脚本从fastq文件中提取sgRNA（small guide RNA）序列，并计算标准化读段计数。