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Efficient CRISPR/Cas9 Genome Editing of Phytoene desaturase in Cassava
Sunday, 2017/11/12 | 05:53:34
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Odipio J, Alicai T, Ingelbrecht I, Nusinow D, Bart R, Taylor JN Front. Plant Sci., 18 October 2017 | https://doi.org/10.3389/fpls.2017.01780
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 5′ and or downstream of the 3′ 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.
See: https://www.frontiersin.org/articles/10.3389/fpls.2017.01780/full
FIGURE 1. Schematic representations of the cassava MePDS target gene, location of the gRNAs, and CRISPR/Cas9 gene-editing construct. (A) Structural organization of the MePDS gene. Exons and introns are shown as boxes and lines, respectively, with the number of exons indicated below boxes. The gRNA where designed from the encircled exon 13. (B) Schematic representation of target region showing the sequences and location of the two 20 bp gRNAs. The sequence shown is a reverse complement of MePDS from cultivar AM560-2 (Phytozome 11, cassava4.1_004359m.g). gRNA1 and gRNA2 are highlighted in yellow and green, respectively, within exon 13 shown as upper case and surrounding intron sequences appears as lower case. Positions of forward (F) and reverse (R) primers flanking the target region in MePDS are indicted with red arrows, respectively. (C) Schematic of the CRISPR/Cas9 binary vector pCAMBIA2300_CR3-EF used for stable Agrobacterium-mediated transformation of cassava. Arabidopsis thaliana promoter (AtU6-26) drives expression of each gRNA used to target MePDS, with the gRNA ligated at the position indicated by the yellow box aided by the Bsa1 restriction site. The Cauliflower mosaic virus promoter (CaMV 35S) drives expression of the Cas9 gene which together with inserted gRNA induce mutations in target region of MePDS gene; NLS, nuclear localization signal; Nos, Nos terminator; LB, left border; RB, right border. Position of forward (F) and reverse (R) primers used for amplifying respective regions of cassette are shown by red arrows. |
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