Efficient CRISPR/Cas9 Genome Editing of Phytoene desaturase in Cassava

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dc.contributor.authorOdipio, John
dc.contributor.authorAlicai, Titus
dc.contributor.authorNusinow, Dmitri
dc.contributor.authorBart, Rebecca
dc.contributor.authorIngelbrecht, Ivan
dc.contributor.authorTaylor, Nigel
dc.date.accessioned2022-06-23T17:46:39Z
dc.date.available2022-06-23T17:46:39Z
dc.date.issued2017
dc.description.abstractCRISPR/Cas9 has become a powerful genome-editing tool for introducing genetic changes into crop species. In order to develop capacity for CRISPR/Cas9 technology in the tropical staple cassava (Manihot esculenta), the Phytoene desaturase (MePDS) gene was targeted in two cultivars using constructs carrying gRNAs targeting two sequences within MePDS exon 13. After Agrobacterium-mediated delivery of CRISPR/Cas9 reagents into cassava cells, both constructs induced visible albino phenotypes within cotyledon-stage somatic embryos regenerating on selection medium and the plants regenerated therefrom. A total of 58 (cv. 60444) and 25 (cv. TME 204) plant lines were recovered, of which 38 plant lines (19 from each cultivar) were analyzed for mutagenesis. The frequency of plant lines showing albino phenotype was high, ranging from 90 to 100% in cv. TME 204. Observed albino phenotypes were comprised of full albinos devoid of green tissue and chimeras containing a mixture of white and green tissues. Sequence analysis revealed that 38/38 (100%) of the plant lines examined carried mutations at the targeted MePDS site, with insertions, deletions, and substitutions recorded. One putatively mono-allelic homozygous line (1/19) was found from cv. 60444, while 1 (1/19) and 4 (4/19) putatively bi-allelic homozygous lines were found in 60444 and TME204, respectively. The remaining plant lines, comprised mostly of the chimeras, were found to be putatively heterozygous. We observed minor (1 bp) nucleotide substitutions and or deletions upstream of the 50 and or downstream of the 30 targeted MePDS region. The data reported demonstrates that CRISPR/Cas9-mediated genome editing of cassava is highly efficient and relatively simple, generating multi-allelic mutations in both cultivars studied. Modification of MePDS described here generates visually detectable mutated events in a relatively short time frame of 6–8 weeks, and does not require sequencing to confirm editing at the target. It therefore provides a valuable platform to facilitate rapid assessment and optimization of CRISPR/Cas9 and other genome-editing technologies in cassava.en_US
dc.identifier.citationOdipio J, Alicai T, Ingelbrecht I, Nusinow DA, Bart R and Taylor NJ (2017) Efficient CRISPR/Cas9 Genome Editing of Phytoene desaturase in Cassava. Front. Plant Sci. 8:1780. doi: 10.3389/fpls.2017.01780en_US
dc.identifier.other10.3389/fpls.2017.01780
dc.identifier.urihttps://nru.uncst.go.ug/handle/123456789/4060
dc.language.isoenen_US
dc.publisherFrontiers in Plant Scienceen_US
dc.subjectCassavaen_US
dc.subjectGenome editingen_US
dc.subjectCRISPR/Cas9en_US
dc.subjectPhytoene desaturase (PDS)en_US
dc.subjectAlbinoen_US
dc.subjectMutationen_US
dc.subjectHomozygousen_US
dc.subjectHeterozygousen_US
dc.titleEfficient CRISPR/Cas9 Genome Editing of Phytoene desaturase in Cassavaen_US
dc.typeArticleen_US
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