There has been no method for performing genome-wide loss-of-function screening in cultured cells until the appearance of two recent papers that described a CRISPR-Cas9-mediated genome-wide knockout screen. We have developed a similar CRISPR-Cas9-based method: using a library containing 77,406 unique single guide RNA (sgRNA) sequences targeting 20,121 genes delivered with a lentiviral vector in combination with transient expression of Cas9, we performed screens to identify the genes essential for HIV-induced cell death. We increased screening specificity by removing the major sources of noise, which is one of the most serious problems in genome-wide functional screening. The results show that our method can identify target genes with high specificity. Here we propose to validate the identified genes and further improve the efficiency of genome-wide screening by improving gRNA library design and screening strategy. In addition, we propose to identify host factors that might not be essential for HIV-induced cell death but contribute to regulation of the process, which will provide systematic and unbiased information about the interaction between HIV and host cell factors.
The proposed research is relevant to public health, because the project aims to develop a new approach to studying the interaction between HIV and host cells to gain systematic insight into HIV-induced cell death, and the results might provide new therapeutic targets for treating HIV infection.
|Ma, Hongming; Dang, Ying; Wu, Yonggan et al. (2015) A CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death. Cell Rep 12:673-83|
|Dang, Ying; Jia, Gengxiang; Choi, Jennie et al. (2015) Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency. Genome Biol 16:280|