High-content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries.

•First use of arrayed, synthetic crRNAs in a multiparametric gene knockout screen.•High-content analysis was used to identify hits in a cell cycle reporter assay.•7 experimental parameters measured in live cells to classify cell into 6 categories.•Hit stratification: target gene expression, indel formation, and gene knockdown.•3 or 4 out of 4 crRNAs confirm the phenotype for most gene target hits.