{"doi":"10.1101/2021.09.08.459297","title":"Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells","abstract":"Abstract Background & Aims 2D monolayers of primary intestinal and colonic epithelial cells represent next-generation in vitro models of the gut. Efficient transgenesis and gene-editing in human intestinal stem cells (hISCs) would significantly improve utility of these models by enabling generation of reporter and loss/gain-of-function hISCs, but no published methods exist for transfecting 2D hISC monolayers. Electroporation has proven effective in other difficult-to-transfect cells; thus we applied this method to hISCs. Methods Twenty-four electroporation parameters were tested, and the optimal condition for efficiency and viability was validated on hISCs from six anatomical regions along the small intestine and colon. PiggyBac™ transposase and Cas9 ribonucleoprotein (RNP) complexes were used for stable genomic integration of reporter genes. High-throughput methods for clone isolation, expansion, and screening were developed. An hISC OLFM4-emGFP reporter was generated and validated by qPCR, organoid assays, and hISC compartmentalization on a planar crypt-microarray (PCM) device. Results Maximum electroporation efficiency was 79.9% with a mean survival of 65%. Transfection of 10 5 hISCs produced ∼142 (0.14%) stable transposase-mediated clones. Transfection of OLFM4 -targetting RNPs yielded ∼35% editing and 99/220 (45%) of antibiotic-resistant colonies analyzed expressed emGFP. OLFM4-emGFP hISCs applied to PCMs remained emGFP+ and proliferative in high-Wnt3a/R-spondin3/Noggin zones yet differentiated to emGFP-/KRT20+ cells outside engineered crypt zones. OLFM4-emGFP levels correlated with endogenous OLFM4 . Olfm4-emGFP high cells were LGR5 high / KRT20 low , and demonstrated high organoid-forming potential. Conclusions Electroporation of hISCs is highly efficient for stable transgenesis and transgenic lines can be generated in 3-4 weeks. Workflows mirror conventional culture methods, facilitating rapid integration into established tissue-culture operations. OLFM4 high is a robust hISC marker with functional properties in culture.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":216093,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9528,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":805775,"name":"Meryem T. Ok","orcid":"0000-0001-6333-8891","position":1,"is_corresponding":false},{"id":805776,"name":"Ismael Gomez-Martinez","orcid":"0000-0002-9445-9642","position":2,"is_corresponding":false},{"id":60544,"name":"Joseph Burclaff","orcid":"0000-0002-0325-5039","position":3,"is_corresponding":false},{"id":811979,"name":"Nathan P. Kohn","orcid":null,"position":4,"is_corresponding":false},{"id":60558,"name":"Scott T. Magness","orcid":"0000-0002-2746-1224","position":5,"is_corresponding":false},{"id":567681,"name":"Keith A. Breau","orcid":"0000-0001-6577-4494","position":0,"is_corresponding":true}],"reference_count":58,"raw_metadata":null,"created_at":"2026-07-18T23:53:00.192353Z","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":[]}