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Background   Neospora caninum  is an obligate intracellular parasite that invades host cells and replicates within the parasitophorous vacuole (PV), which resists fusion with host cell lysosomal compartments. To modify the PV, the parasite secretes an array of proteins, including dense granule proteins (GRAs). The vital role of GRAs in the  Neospora  life cycle cannot be overestimated. Despite this important role, only a subset of these proteins have been identified, and most of their functions have not been elucidated. Our previous study demonstrated that NcGRA17 is specifically targeted to the delimiting membrane of the parasitophorous vacuole membrane (PVM). In this study, we utilize proximity-dependent biotin identification (BioID) to identify novel components of the dense granules.    Methods  NcGRA17 was BirA* epitope-tagged in the Nc1 strain utilizing the CRISPR/Cas9 system to create a fusion of NcGRA17 with the biotin ligase BirA*. The biotinylated proteins were affinity-purified for mass spectrometric analysis, and the candidate GRA proteins from BioID data set were identified by gene tagging. To verify the biological role of novel identified GRA proteins, we constructed the  NcGRA23  and  NcGRA11 (a–e)  knockout strains using the CRISPR/Cas9 system and analyzed the phenotypes of these mutants.    Results  Using NcGRA17-BirA* fusion protein as bait, we have identified some known GRAs and verified localization of 11 novel GRA proteins by gene endogenous tagging or overexpression in the Nc1 strain. We proceeded to functionally characterize NcGRA23 and NcGRA11 (a–e) by gene knockout. The lack of NcGRA23 or NcGRA11 (a–e) did not affect the parasite propagation in vitro and virulence in vivo.    Conclusions  In summary, our findings reveal that BioID is effective in discovering novel constituents of  N. caninum  dense granules. The exact biological functions of the novel GRA proteins are yet unknown, but this could be explored in future studies.    Graphical abstractSupplementary Information  The online version contains supplementary material available at 10.1186/s13071-021-05023-7.

Biotinylation of the Neospora caninum parasitophorous vacuole reveals novel dense granule proteins