Genome-wide functional genomic screens utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system have proven to be a powerful tool for systematic genomic perturbation in mammalian cells and provide an alternative to previous screens utilizing RNA interference technology. The wide availability of these libraries through public plasmid repositories as well as the decreasing cost and speed in quantifying these screens using high-throughput next-generation sequencing (NGS) allows for the adoption of the technology in a variety of laboratories interested in diverse biologic questions. Here, we describe the protocol to generate next-generation sequencing libraries from genome-wide CRISPR genomic screens.

Targeting mutant KRAS signaling pathways continues to attract attention as a therapeutic strategy for KRAS-driven tumors. In this study, we exploited the power of the CRISPR-Cas9 system to identify genes affecting the tumor xenograft growth of human mutant KRAS (KRAS^MUT) colorectal cancers. Using pooled lentiviral single-guide RNA libraries, we conducted a genome-wide loss-of-function genetic screen in an isogenic pair of human colorectal cancer cell lines harboring mutant or wild-type KRAS. The screen identified novel and established synthetic enhancers or synthetic lethals for KRAS^MUT colorectal cancer, including targetable metabolic genes. Notably, genetic disruption or pharmacologic inhibition of the metabolic enzymes NAD kinase or ketohexokinase was growth inhibitory in vivo In addition, the chromatin remodeling protein INO80C was identified as a novel tumor suppressor in KRAS^MUT colorectal and pancreatic tumor xenografts. Our findings define a novel targetable set of therapeutic targets for KRAS^MUT tumors. Cancer Res; 77(22); 6330-9. ©2017 AACR.

We perform a CRISPR-Cas9 genome-wide screen in glioblastoma stem cells and identify integrin αvβ5 as an internalization factor for Zika virus (ZIKV). Expression of αvβ5 is correlated with ZIKV susceptibility in various cells and tropism in developing human cerebral cortex. A blocking antibody against integrin αvβ5, but not αvβ3, efficiently inhibits ZIKV infection. ZIKV binds to cells but fails to internalize when treated with integrin αvβ5-blocking antibody. αvβ5 directly binds to ZIKV virions and activates focal adhesion kinase, which is required for ZIKV infection. Finally, αvβ5 blocking antibody or two inhibitors, SB273005 and cilengitide, reduces ZIKV infection and alleviates ZIKV-induced pathology in human neural stem cells and in mouse brain. Altogether, our findings identify integrin αvβ5 as an internalization factor for ZIKV, providing a promising therapeutic target, as well as two drug candidates for prophylactic use or treatments for ZIKV infections.