Efficient CRISPR/Cas9 Genome Editing of Phytoene desaturase in Cassava
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Date
2017
Journal Title
Journal ISSN
Volume Title
Publisher
Frontiers in Plant Science
Abstract
CRISPR/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.
Description
Keywords
Cassava, Genome editing, CRISPR/Cas9, Phytoene desaturase (PDS), Albino, Mutation, Homozygous, Heterozygous
Citation
Odipio 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.2017.01780