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dc.contributor.authorSubburaj, Saminathan
dc.contributor.authorZanatta, Caroline Bedin
dc.contributor.authorNunn, Jennifer Ann Lillebo
dc.contributor.authorHoepers, Aline Martins
dc.contributor.authorNodari, Rubens Onofre
dc.contributor.authorAgapito-Tenfen, Sarah Zanon
dc.date.accessioned2022-11-09T09:05:20Z
dc.date.available2022-11-09T09:05:20Z
dc.date.issued2022-07-12
dc.description.abstractCRISPR/Cas9-based ribonucleoprotein (RNP)-mediated system has the property of minimizing the effects related to the unwanted introduction of vector DNA and random integration of recombinant DNA. Here, we describe a platform based on the direct delivery of Cas9 RNPs to soybean protoplasts for genetic screens in knockout gene-edited soybean lines without the transfection of DNA vectors. The platform is based on the isolation of soybean protoplasts and delivery of Cas RNP complex. To empirically test our platform, we have chosen a model gene from the soybean genetic toolbox. We have used five different guide RNA (gRNA) sequences that targeted the constitutive pathogen response 5 (CPR5) gene associated with the growth of trichomes in soybean. In addition, efficient protoplast transformation, concentration, and ratio of Cas9 and gRNAs were optimized for soybean for the first time. Targeted mutagenesis insertion and deletion frequency and sequences were analyzed using both Sanger and targeted deep sequencing strategies. We were able to identify different mutation patterns within insertions and deletions (InDels) between + 5 nt and –30 bp and mutation frequency ranging from 4.2 to 18.1% in the GmCPR5 locus. Our results showed that DNA-free delivery of Cas9 complexes to protoplasts is a useful approach to perform early-stage genetic screens and anticipated analysis of Cas9 activity in soybeans.en_US
dc.identifier.citationSubburaj S, Zanatta CB, Nunn JAl, Hoepers, Nodari RO, Agapito-Tenfen SZ. A DNA-Free Editing Platform for Genetic Screens in Soybean via CRISPR/Cas9 Ribonucleoprotein Delivery. Frontiers in Plant Science. 2022;13en_US
dc.identifier.cristinIDFRIDAID 2045553
dc.identifier.doi10.3389/fpls.2022.939997
dc.identifier.issn1664-462X
dc.identifier.urihttps://hdl.handle.net/10037/27308
dc.language.isoengen_US
dc.publisherFrontiers Mediaen_US
dc.relation.journalFrontiers in Plant Science
dc.rights.accessRightsopenAccessen_US
dc.rights.holderCopyright 2022 The Author(s)en_US
dc.rights.urihttps://creativecommons.org/licenses/by/4.0en_US
dc.rightsAttribution 4.0 International (CC BY 4.0)en_US
dc.titleA DNA-Free Editing Platform for Genetic Screens in Soybean via CRISPR/Cas9 Ribonucleoprotein Deliveryen_US
dc.type.versionpublishedVersionen_US
dc.typeJournal articleen_US
dc.typeTidsskriftartikkelen_US
dc.typePeer revieweden_US


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Attribution 4.0 International (CC BY 4.0)
Med mindre det står noe annet, er denne innførselens lisens beskrevet som Attribution 4.0 International (CC BY 4.0)