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The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen2 1-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG2 1-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG2 11 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG2 1-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications. [Display omitted] • The split-mNG2 system achieves tissue-specific protein labeling while maintaining endogenous expression levels. • mNG2 1-10 is expressed under a tissue-specific promoter using standard transgenesis. • mNG2 11 is fused to proteins of interest using CRISPR/Cas-directed gene editing. • The feasibility of the split-mNG2 approach is demonstrated for the cytoskeletal genes tubb4b and krt8. • The split-mNG2 system can also be used to direct protein localization. [ABSTRACT FROM AUTHOR]