Genome-scale CRISPR screen reveals inhibitory factors as regulatory module of HIV

To identify host factors that restrict HIV replication, we conducted a CRISPR activation screen in a susceptible T cell line using a high-complexity, genome-wide sgRNA library. Our results identified host factors that conferred protection from HIV infection. To identify cellular factors restricting HIV-1 replication, we utilized a previously engineered synergistic activation mediator complex (SAM) for the transcriptional activation of endogenous genes. dCas9 and MS2 expressing CD4 T cells were expanded and 300 million cells were further transduced with the SAM lentiviral library (3 sgRNAs per gene for 70,290 guides in total) at an MOI = 0.3 for 7 days. The gain-of-expression cells were divided into two pools. We challenged the first pool of 60 million transduced cell with HIV at an MOI of 1 for 4 days and then treated cells with GCV for 3 days to further deplete infected cells. Two rounds of infection and GCV selection were performed to enrich HIV resistant cells to identify host genes important in HIV infection. The other pool of 30 million cells were mock infected as control. After 2 weeks, surviving cells were sorted out. Genomic DNA was extracted from harvested cells and sgRNAs were PCR-amplified and deep sequenced. Normalized reads of HIV infected cells were compared to mock infected cells.

为鉴定限制HIV复制的宿主因子，我们采用高复杂度全基因组单引导RNA（single-guide RNA, sgRNA）文库，在易感T细胞系中开展CRISPR激活筛选，最终鉴定出可赋予细胞抵御HIV感染能力的宿主因子。为进一步鉴定限制HIV-1复制的细胞因子，我们借助此前构建的协同激活介导复合物（synergistic activation mediator, SAM）对内源基因进行转录激活。将表达dCas9与MS2的CD4阳性T细胞扩增培养后，取3亿个细胞以感染复数（multiplicity of infection, MOI）=0.3的比例，使用SAM慢病毒文库（每个基因对应3条sgRNA，总计70290条向导序列）进行转导，持续培养7天。将基因表达上调的细胞分为两个细胞池：我们以MOI=1的感染量用HIV攻毒培养第一池中的6000万个转导细胞4天，随后用更昔洛韦（ganciclovir, GCV）处理细胞3天以进一步清除感染细胞，通过两轮感染与更昔洛韦筛选富集获得HIV抗性细胞，以此鉴定与HIV感染相关的宿主基因；另一池的3000万个细胞则以模拟感染作为对照。两周后分离收集存活细胞，从收集的细胞中提取基因组DNA，通过PCR扩增sgRNA序列并进行深度测序，将HIV感染组细胞的归一化读段数与模拟感染对照组进行比对分析。