Fig 1.

Genome-wide guide RNA (gRNA) library screening of essential factors of rAAV2.5T transduction in HeLa S3 cells. (A) A diagram of the genome-wide gRNA library screening. HeLa S3 suspension cells were transduced with a spCas9-expressing lentivirus. Blasticidin-resistant spCas9-expressing cells were transduced with the Brunello lentiCRISPR gRNA lentiviral library. The library-selected HeLa S3 cells were expanded to ~200 million, of which ~100 million were extracted for genomic DNA (gDNA) as the non-selected control gDNA (gDNA^Ctrl), and another 100 million were transduced with mCherry-expressing rAAV2.5T. At 3 days post-transduction, ~1 million mCherry-negative cells were sorted and expanded. Approximately 100 million cells were transduced with rAAV2.5T, followed by sorting of the top 1% mCherry-negative cells. The sorted mCherry-negative cells were expanded to ~100 million for extraction of gDNA^Screen. The gDNA samples were subjected to NGS analysis and bioinformatics analysis. Created with BioRender. (B) Hits of host genes from the second round of screening of mCherry-negative cells. This panel shows the entry screening hits from the second-round selection of mCherry-negative cells transduced with rAAV2.5T. The x-axis shows genes within the target of the Brunello library that are grouped by gene ontology analysis. Each circle represents a gene with the size corresponding to the fold change of sgRNA reads by comparison of gDNA^Screen to gDNA^Ctrl. The y-axis shows the enrichment score (−log₁₀) of each gene based on MAGeCK analysis of the sgRNA reads in gDNA^Screen vs gDNA^Ctrl. Only genes that have an enrichment score of >3.25 are shown. The bubble colors indicate different gene ontology classifications as shown.