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Ablation of OCT4 function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART)

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dc.contributor.authorNix, Jada L.en
dc.contributor.authorSchettini, Gustavo P.en
dc.contributor.authorSpeckhart, Savannah L.en
dc.contributor.authorEaly, Alan D.en
dc.contributor.authorBiase, Fernando H.en
dc.date.accessioned2024-01-23T14:40:39Zen
dc.date.available2024-01-23T14:40:39Zen
dc.date.issued2023-11-01en
dc.description.abstractCRISPR-Cas ribonucleoproteins (RNPs) are important tools for gene editing in preimplantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the presence of maternal RNAs that support embryo development until embryonic genome activation may cause confounding phenotypes. Here, we aimed to improve the efficiency of biallelic deletions and deplete specific maternal RNAs in cattle zygotes using CRISPR-Cas editing technology. Two electroporation sessions with Cas9D10A RNPs targeting exon 1 and the promoter of OCT4 produced biallelic deletions in 91% of the embryos tested. In most cases, the deletions were longer than 1,000 nucleotides long. Electroporation of Cas13a RNPs prevents the production of the corresponding proteins. We electroporated Cas9D10A RNPs targeting exon 1, including the promoter region, of OCT4 in two sessions with inclusion of Cas13a RNPs targeting OCT4 mRNAs in the second session to ablate OCT4 function in cattle embryos. A lack of OCT4 resulted in embryos arresting development prior to blastocyst formation at a greater proportion (13%) than controls (31.6%, P < 0.001). The few embryos that developed past the morula stage did not form a normal inner cell mass. Transcriptome analysis of single blastocysts, confirmed to lack exon 1 and promoter region of OCT4, revealed a significant (False Discovery Rate, FDR < 0.1) reduction in transcript abundance of many genes functionally connected to stemness, including markers of pluripotency (CADHD1, DPPA4, GNL3, RRM2). The results confirm that OCT4 is a key regulator of genes that modulate pluripotency and is required to form a functional blastocyst in cattle.en
dc.description.versionPublished versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1093/pnasnexus/pgad343en
dc.identifier.eissn2752-6542en
dc.identifier.issn2752-6542en
dc.identifier.issue11en
dc.identifier.orcidBiase, Fernando [0000-0001-7895-0223]en
dc.identifier.orcidEaly, Alan [0000-0002-8507-6578]en
dc.identifier.otherPMC10637268en
dc.identifier.otherpgad343 (PII)en
dc.identifier.pmid37954164en
dc.identifier.urihttps://hdl.handle.net/10919/117605en
dc.identifier.volume2en
dc.language.isoenen
dc.publisherOxford University Pressen
dc.relation.urihttps://www.ncbi.nlm.nih.gov/pubmed/37954164en
dc.rightsCreative Commons Attribution-NonCommercial-NoDerivatives 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/en
dc.subjectCRISPRen
dc.subjectelectroporationen
dc.subjectgene editingen
dc.subjectpreimplantation developmenten
dc.titleAblation of OCT4 function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART)en
dc.title.serialPNAS Nexusen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
dc.type.otherJournal Articleen
dcterms.dateAccepted2023-10-11en
pubs.organisational-group/Virginia Techen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciencesen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/School of Animal Sciencesen
pubs.organisational-group/Virginia Tech/Faculty of Health Sciencesen
pubs.organisational-group/Virginia Tech/All T&R Facultyen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/CALS T&R Facultyen

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