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To overcome this limitation, we performed yeast surface display\u2010based directed evolution to enhance APEX2 activity toward Btn\u2010An. The resulting variant,\n                    <jats:sup>L242F<\/jats:sup>\n                    APEX2, exhibits an approximately two\u2010fold improvement in labeling efficiency, likely through enhanced enzyme\u2010substrate interactions. This increased activity enables rapid and efficient proximity labeling while maintaining spatial specificity. After validating its performance at the ER membrane, we applied\n                    <jats:sup>L242F<\/jats:sup>\n                    APEX2 to profile the transcriptome proximal to the midbody and identified\n                    <jats:italic>ANLN<\/jats:italic>\n                    as a previously unreported midbody\u2010localized mRNA during telophase. Drug perturbation and reporter assays further revealed that\n                    <jats:italic>ANLN<\/jats:italic>\n                    mRNA targeting to the midbody occurs co\u2010translationally and depends on its nascent N\u2010terminal peptide. Together, this work establishes\n                    <jats:sup>L242F<\/jats:sup>\n                    APEX2 as an improved and versatile tool for spatially resolved transcriptomics in complex subcellular contexts.\n                  <\/jats:p>","DOI":"10.1002\/advs.75012","type":"journal-article","created":{"date-parts":[[2026,3,28]],"date-time":"2026-03-28T16:43:33Z","timestamp":1774716213000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Directed Evolution Improves the Catalytic Efficiency of APEX2\u2010Mediated Proximity\u2010Dependent RNA Labeling"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-7799-0196","authenticated-orcid":false,"given":"Gang","family":"Wang","sequence":"first","affiliation":[{"name":"College of Chemistry and Molecular Engineering Synthetic and Functional Biomolecules Center Beijing National Laboratory For Molecular Sciences PKU\u2010IDG\/McGovern Institute for Brain Research Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education Beijing Advanced Center of RNA Biology (BEACON), Peking University Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Li","sequence":"additional","affiliation":[{"name":"College of Chemistry and Molecular Engineering Synthetic and Functional Biomolecules Center Beijing National Laboratory For Molecular Sciences PKU\u2010IDG\/McGovern Institute for Brain Research Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education Beijing Advanced Center of RNA Biology (BEACON), Peking University Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peiyuan","family":"Meng","sequence":"additional","affiliation":[{"name":"College of Chemistry and Molecular Engineering Synthetic and Functional Biomolecules Center Beijing National Laboratory For Molecular Sciences PKU\u2010IDG\/McGovern 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