Frequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryos

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dc.contributor.authorCarey, Kaylaen
dc.contributor.authorRyu, Junghyunen
dc.contributor.authorUh, Kyungjunen
dc.contributor.authorLengi, Andrea J.en
dc.contributor.authorClark-Deener, Sherrieen
dc.contributor.authorCorl, Benjamin A.en
dc.contributor.authorLee, Kihoen
dc.contributor.departmentAnimal and Poultry Sciencesen
dc.contributor.departmentDairy Scienceen
dc.contributor.departmentLarge Animal Clinical Sciencesen
dc.date.accessioned2019-05-13T11:47:03Zen
dc.date.available2019-05-13T11:47:03Zen
dc.date.issued2019-05-06en
dc.date.updated2019-05-12T20:14:20Zen
dc.description.abstractBackground The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined. Results First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5′ side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5′ side, indicating unpredictability of the events. Conclusions The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.en
dc.description.versionPublished versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.citationBMC Biotechnology. 2019 May 06;19(1):25en
dc.identifier.doihttps://doi.org/10.1186/s12896-019-0517-7en
dc.identifier.urihttp://hdl.handle.net/10919/89491en
dc.language.isoenen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.holderThe Author(s)en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.titleFrequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryosen
dc.title.serialBMC Biotechnologyen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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