Amplicon Sequencing of gRNAs from Brunello CRISPR mutagenized A549 and U87-MG cells after live/dead selection with EV-D68 IL

With the near eradication of poliovirus due to global vaccination campaigns, attention has shifted to other enteroviruses that can cause polio-like paralysis syndrome (now termed acute flaccid myelitis (AFM)). In particular, enterovirus D68 (EV-D68) is believed to be the main driver of epidemic outbreaks of AFM in recent years, yet not much is known about EV-D68 host interactions. EV-D68 is a respiratory virus but, in rare cases, can spread to the central nervous system to cause severe neuropathogenesis. We use genome-scale CRISPR screens to identify genes important for EV-D68 infection. A549 and U87-MG cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, 300 million cells that constitutively express Cas9 were transduced with lentiGuide-Puro from the Brunello library (MOI 0.3). Cells were then selected with puromycin, expanded to 3 billion cells, and then pooled together and cryofrozen in aliquots. One hundred million cells were thawed constituting over 1000× genome coverage worth of mutagenized library,expanded, and seeded for the screens. Each screen had over 500x genome coverage. The cells were infected with EV-D68 IL (BEI USA/2014/18952) (MOI 0.1). Virus-resistant colonies were harvested. The uninfected reference used was the unselected starting population. The unselected and selected cells were both processed with QIAamp DNA columns to purify the gDNA. A first round of PCR was used to amplify the guide RNA sequences encoded in the gDNA, followed by a second round of PCR to add the barcodes/adapters for amplicon sequencing. 2% agarose gels and a QIAquick gel extraction kit were used to purify the amplicons. The amplicons were then subjected to next-generation sequencing on a HiSeq instrument lane (Illumina) via Novogene.