Pooled CRISPR screens have been widely applied to mammalian and other organisms to elucidate the interplay between genes and phenotypes of interest. The most popular method for delivering the CRISPR components into mammalian cells is lentivirus based. However, lentivirus is not always an option, virus-free (VF) protocols are starting to emerge. Here we demonstrate an improved virus-free (VF) genome-wide CRISPR screening platform for Chinese hamster ovary (CHO) cells with 75,488 gRNAs targeting 15,028 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad, resulting in a very high percentage of single gRNA insertion and minimal clonal variation. Using this platform, we perform a negative selection screen on cell proliferation that identifies 1980 genes that affect proliferation in CHO and a positive selection screen on toxic endoplasmic reticulum (ER) stress inducer, tunicamycin, that identifies 77 gene KOs that improve survivability.