Orthogonal genome-wide screenings in bat cells identify MTHFD1 as a target of broad antiviral therapy

Danielle E Anderson; Jin Cui; Qian Ye; Baoying Huang; Wenhong Zu; Jing Gong; Weiqiang Liu; So Young Kim; Biao Guo Yan; Kristmundur Sigmundsson; Xiao Fang Lim; Fei Ye; Peihua Niu; Xuming Zhou; Wenjie Tan; Lin-Fa Wang; Xu Tan

infections. For example, the DNA damage checkpoint pathway and the NF-kB pathway are 25 known to be positively selected in the bat genomes 9 . In addition, in the type I interferon gene 26 family, the MHC class I genes and natural killer cell receptor family have been reported to be 27 highly expanded in the bat genomes 10 . These studies provided evidence to support the hypothesis 28 that bats have evolved a highly specific genetic configuration, especially in the innate immunity 29 pathways. A major caveat is the lack of systematic examination of the functions of these genes 30 due to shortage of functional genomic tools. 31 CRISPR and RNAi technologies have enabled genome-wide screening of gene functions for a 1 variety of cell types, yielding an unprecedented wealth of information revealing the complexity of 2 biological processes 11, 12 . This is especially true in the field of virus-host interactions, where a 3 plethora of host factors have been identified for a variety of viruses [13] [14] [15] . Both technologies have 4 advantages and disadvantages and provide complementary approaches to achieve comprehensive 5 genetic perturbations [16] [17] [18] . CRISPR permits complete knockout of a gene, thus affords generation 6 of truly null genotype. A caveat is that the lethality associated with knockout of essential genes 7 prevents functional parsing of these genes in different biological processes. In addition, CRISPR 8 screening is usually used a pool-based format that requires the generation of stable cell lines 9 through long term culturing. Therefore, CRISPR is suited for studying phenotypes that require a 10 longer time to manifest. Based on previous studies, CRISPR screens demonstrated excellent 11 specificity but limited sensitivity 17 . In contrast, siRNA screening relies on temporary knockdown 12 of genes in an individual well format, a methodology suited for examining short-term phenotypic 13 changes. Due to the transient nature of the assay, siRNA screening can tolerate growth defects due 14 to gene knockdown and can elucidate hypomorphic phenotypes induced by siRNAs with varying 15 degrees of knockdown. In general, RNAi screens allow comprehensive identification of genes in 16 a biological process at the cost of more false positives due to pervasive off-target effects 17 . To fully 17 realize the power of CRISPR and RNAi screening as complementary strategies to interrogate gene 18 function in bats, here we developed a pool-based CRISPR library and a plate-based siRNA library 19