Tissue-specific and endogenous protein labeling with split fluorescent proteins

Abstract

AbstractThe 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 mNeonGreen21-10/11(split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10is expressed under a tissue-specific promoter using standard transgenesis while mNG211is 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 genestubb4bandkrt8in various tissues. We also demonstrate that by anchoring the mNG21-10component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein function. Our approach should be broadly useful for a wide range of applications.