Efficient CRISPR/Cas9 Genome Editing of Phytoene desaturase in Cassava

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 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.

Simultaneous CRISPR/Cas9‐mediated editing of cassava eIF4E isoforms nCBP‐1 and nCBP‐2 reduces cassava brown streak disease symptom severity and incidence

“…Integrating CRISPR/Cas9 constructs into the cassava genome proved to be efficient for achieving gene editing as 91% of transformed lines carried INDELs at the target sites, and desired homozygous and bi‐allelic mutations were observed in 78% of plant lines. These frequencies compare favourably to previous CRISPR/Cas9‐mediated mutagenesis studies in cassava, rice and tomato (Ma et al ., ; Odipio et al ., ; Pan et al ., ).…”

“…Considering the low incidence of the latter in plants, identical NHEJ repair may be more likely (Peng et al, 2016). While transgenic plants derived from FECs are thought to be of single cell origin ( Figure S3), reducing the likelihood of transgenic chimeras (Schreuder et al, 2001;Taylor et al, 1996), Odipio et al (2017) have reported the production of chimeric plants via CRISPR/Cas9-mediated gene editing of phytoene desaturase in cassava. Complex genotypes were not characterized in depth, but they are likely chimeras resulting from Cas9/ sgRNA activity being delayed until after embryogenic units began to replicate (Odipio et al, 2017;Zhang et al, 2014).…”

“…While transgenic plants derived from FECs are thought to be of single cell origin ( Figure S3), reducing the likelihood of transgenic chimeras (Schreuder et al, 2001;Taylor et al, 1996), Odipio et al (2017) have reported the production of chimeric plants via CRISPR/Cas9-mediated gene editing of phytoene desaturase in cassava. Complex genotypes were not characterized in depth, but they are likely chimeras resulting from Cas9/ sgRNA activity being delayed until after embryogenic units began to replicate (Odipio et al, 2017;Zhang et al, 2014). Integrating CRISPR/Cas9 constructs into the cassava genome proved to be efficient for achieving gene editing as 91% of transformed lines carried INDELs at the target sites, and desired homozygous and bi-allelic mutations were observed in 78% of plant lines.…”

“…mutagenized allele and one wild-type allele. Plants were considered complex if they carried more than two sequence patterns, strongly suggesting chimerism (Odipio et al, 2017;Zhang et al, 2014). The genotypes of edited plants had Cas9-induced INDELs ranging from insertions of 1-16 bp and deletions as large as 127 bp (Table S1).…”

Production of very-high-amylose cassava by post-transcriptional silencing of branching enzyme genes

“…Compared with other starches (e.g., from maize and potato), pure-white cassava starch contains low levels of fat (0.08-1.54%), proteins (0.03-0.6%), and phosphorus (0.75-4%), making it an excellent starch resource for various industrial applications (Richard et al, 1991;Moorthy, 2002;Balagopalan, 2002;Sharma et al, 2016). Modified starches of cassava have been broadly used in the food, textile, Wang et al, 2014;Bredeson et al, 2016) and development of cassava transformation technology (Liu et al, 2011;Zhang et al, 2017), we now have the capacity to breed novel cassava cultivars with desirable traits using state-of-the-art technologies including CRISPR/Cas9-based genome editing (Odipio et al, 2017;Sun et al, 2017;Bull et al, 2018;Gomez et al, 2018).…”