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Our first step in constructing an optogenetic system to regulate endogenous Drosophila gene expression was to identify inhibitory anti-CRISPR (Acr) proteins that block CRISPRa-mediated activation. Next, we inserted optogenetic protein LOV2 into these Acrs, tested for their ability to optogenetically modulate endogenous gene upregulation through the CRISPRa-based flySAM system in Drosophila, and found that the photoswitchability of these prototypes was weak. We therefore engineered an optimized Acr\u2013LOV2 fusion module by refining length of intrinsically disordered and ordered regions (IDR and IOR) of Acrs. This optimization yielded a variant with significantly greater sensitivity to blue-light-induced endogenous gene upregulation than the prototypes, leading to new in vivo discoveries. In addition, this work provides insights for in vivo functional characterization of the IDR and the IOR of these small-sized proteins. Together, these findings establish a robust optogenetic toolbox for precise, light-controlled endogenous gene regulation in Drosophila.<\/jats:p>","DOI":"10.1093\/nar\/gkag244","type":"journal-article","created":{"date-parts":[[2026,5,4]],"date-time":"2026-05-04T11:16:49Z","timestamp":1777893409000},"source":"Crossref","is-referenced-by-count":0,"title":["Optimized optogenetic anti-CRISPR for endogenous gene regulation in\n                    <i>Drosophila<\/i>"],"prefix":"10.1093","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3048-5120","authenticated-orcid":false,"given":"Clarissien","family":"Ramongolalaina","sequence":"first","affiliation":[{"name":"Aging and Regeneration Center, School of Basic Medical Sciences, Tsinghua Medicine, Medical Science Building, Room D204, Tsinghua University , Beijing 100084 ,","place":["China"]},{"name":"The State Key Laboratory for Complex, Severe, and Rare Diseases , Beijing 100084 ,","place":["China"]},{"name":"School of Life 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