{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,5]],"date-time":"2026-05-05T00:23:55Z","timestamp":1777940635253,"version":"3.51.4"},"reference-count":18,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T00:00:00Z","timestamp":1777593600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T00:00:00Z","timestamp":1777593600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["febs.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["FEBS Open Bio"],"abstract":"<jats:p>Optical pooled CRISPR screens have become an attractive tool for the rapid identification of genes involved in biological processes. In such screens, mixed populations of cells, each with a single gene knocked out, are screened by microscopy for phenotypes of interest. Identified hit cells can then be tagged by photoactivation of a co\u2010expressed marker, such as PA\u2010mCherry, and subsequently isolated by FACS to identify the responsible guide RNA by next\u2010generation sequencing. Photoactivation is typically performed by selective irradiation of cells with UV light, using either a digital mirror device (DMD), an external fixed UV laser, or, conveniently, by using the 405\u2009nm laser line present in most confocal scanning microscopes. In this study, the latter approach is optimized for PA\u2010mCherry, a bright red phototag used by us and others in optical pooled screens. We find that although normal scanning with intense 405\u2009nm light can rapidly activate PA\u2010mCherry, it also leads to rapid photobleaching. Instead, much higher cellular brightness is achieved by limiting intensity and pixel dwell time during scanning, as well as by slightly defocusing the laser. These results should help optimize cell tagging for genotype\u2013phenotype mapping in optical pooled screens, as well as for other applications.<\/jats:p>","DOI":"10.1002\/2211-5463.70249","type":"journal-article","created":{"date-parts":[[2026,5,2]],"date-time":"2026-05-02T05:56:14Z","timestamp":1777701374000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimizing photoactivation of\n                    <scp>PA<\/scp>\n                    \u2010\n                    <scp>mCherry<\/scp>\n                    for optical pooled\n                    <scp>CRISPR<\/scp>\n                    screens"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4507-8509","authenticated-orcid":false,"given":"Sravasti","family":"Mukherjee","sequence":"first","affiliation":[{"name":"Division of Cell Biology The Netherlands Cancer Institute  Amsterdam The Netherlands"},{"name":"Swammerdam Institute of Life Sciences University of Amsterdam  The Netherlands"},{"name":"Van Leeuwenhoek Centre for Advanced Microscopy (LCAM)  Amsterdam The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-1526-6060","authenticated-orcid":false,"given":"Giulia","family":"Zanetti","sequence":"additional","affiliation":[{"name":"Division of Cell Biology The Netherlands Cancer Institute  Amsterdam The Netherlands"},{"name":"Van Leeuwenhoek Centre for Advanced Microscopy (LCAM)  Amsterdam The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1037-907X","authenticated-orcid":false,"given":"Bram","family":"van den Broek","sequence":"additional","affiliation":[{"name":"Division of Cell Biology The Netherlands Cancer Institute  Amsterdam The Netherlands"},{"name":"Van Leeuwenhoek Centre for Advanced Microscopy (LCAM)  Amsterdam The Netherlands"},{"name":"Bioimaging Facility The Netherlands Cancer Institute  Amsterdam The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7019-3440","authenticated-orcid":false,"given":"Kees","family":"Jalink","sequence":"additional","affiliation":[{"name":"Division of Cell Biology The Netherlands Cancer Institute  Amsterdam The Netherlands"},{"name":"Swammerdam Institute of Life Sciences University of Amsterdam  The Netherlands"},{"name":"Van Leeuwenhoek Centre for Advanced Microscopy (LCAM)  Amsterdam The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2026,5,1]]},"reference":[{"key":"e_1_2_12_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cbpa.2014.05.016"},{"key":"e_1_2_12_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1298"},{"key":"e_1_2_12_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41580\u2010019\u20100186\u20103"},{"key":"e_1_2_12_5_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0032888"},{"key":"e_1_2_12_6_1","doi-asserted-by":"publisher","DOI":"10.1007\/978\u20101\u20104939\u20100470\u20109_14"},{"key":"e_1_2_12_7_1","doi-asserted-by":"publisher","DOI":"10.1091\/mbc.E20\u201010\u20100669"},{"key":"e_1_2_12_8_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.202006180"},{"key":"e_1_2_12_9_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.202008158"},{"key":"e_1_2_12_10_1","doi-asserted-by":"publisher","DOI":"10.15252\/msb.20209442"},{"key":"e_1_2_12_11_1","doi-asserted-by":"crossref","unstructured":"MukherjeeS vanToorenM ZanettiG SfakianakisD KempsD KlarenbeekJ KuikenHJ LieftinkC van denBroekB BeijersbergenRLet\u00a0al. (2025)Beyond static screens: a high\u2010throughput pooled imaging CRISPR platform for dynamic phenotype discovery. 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