{"doi":"10.1101/2022.02.02.478837","title":"Breasi-CRISPR: an efficient genome editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex","abstract":"Abstract In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. This can be challenging when the researched protein lacks reliable antibodies. Here, we combine Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. This method is called Breasi-CRISPR (Brain Easi-CRISPR), and enables knock-in of both short and long epitope tag sequences in genes of interest with high efficiency. Using Breasi-CRISPR, we were able to visualize epitope tagged proteins known to have either high or low expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1, and RPL22. Detection was possible by immunohistochemistry as soon as one day after electroporation at embryonic day 13 (E13). Two and five days after electroporation, we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices two days after electroporation. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores in an efficient manner, allowing the visualization of endogenous proteins via live-imaging in organotypic brain slices two days after electroporation. Finally, we used Breasi-CRISPR to perform co-IP mass-spectrometry analyses of tagged autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data show Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":299773,"datarank":0.20794415416798362,"base_score":1.3862943611198906,"endowment":1.3862943611198906,"self_citation_contribution":0.20794415416798362,"citation_network_contribution":0.0,"self_endowment_contribution":0.20794415416798362,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9479,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":990427,"name":"K C Pratiksha","orcid":null,"position":1,"is_corresponding":false},{"id":371511,"name":"Neeraj K. Tiwari","orcid":"0000-0002-3496-5148","position":2,"is_corresponding":false},{"id":935904,"name":"Claire Kittock","orcid":"0000-0002-0554-0554","position":3,"is_corresponding":false},{"id":913245,"name":"A K Klein","orcid":"0009-0000-5168-3962","position":4,"is_corresponding":false},{"id":475885,"name":"Claire Evans","orcid":null,"position":5,"is_corresponding":false},{"id":371512,"name":"Louis‐Jan Pilaz","orcid":"0000-0002-9035-1327","position":6,"is_corresponding":false},{"id":371510,"name":"Brandon L. Meyerink","orcid":"0000-0002-4058-1685","position":0,"is_corresponding":true}],"reference_count":29,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T00:31:49.412501Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}