Optical enrichment enables accurate sgRNA identification in a pooled CRISPR screen. (a) Schematic of mIFP proof-of-principle screen. A mixed population of mIFP-positive and -negative cells was imaged and photoactivated as described in Fig. 2 c. mCherry-positive and unanalyzed cells were then isolated by FACS and prepared for high-throughput sequencing to extract sgRNA information. (b) Screening result presented by distribution of phenotypic scores of all the sgRNA groups. Red and gray, mIFP groups and Ctrl groups calculated by comparing with unanalyzed sample. (c) Schematic of dual-activation experiment. Experiment as described in Fig. 3 a, but mIFP-negative cells were also photoactivated (100 ms). mIFP-positive cells were activated using a longer activation time (2,000 ms) to guarantee a clear distinction by FACS. Image acquisition generally does not cover the complete imaging well, which leaves cells not imaged and unanalyzed. Lower panel shows an example of FACS data. Cells sorted for mIFP expression (sorted mIFP-positive), cells sorted for lack of mIFP (sorted mIFP-negative), and unanalyzed cells were separately collected and prepared for high-throughput sequencing. (d) Distribution of phenotypic scores of all the sgRNA groups compared with the sorted mIFP-negative sample. Phenotype identification is improved by comparing with true negative cells rather than unanalyzed cells as shown in Fig. 2 b. Red and gray, mIFP groups and Ctrl groups calculated by comparing with sorted mIFP negative sample.