Efficient CRISPR/Cas9 Genome Editing of Phytoene desaturase in Cassava.

John Odipio,Ivan Ingelbrecht,Nigel J Taylor,Dmitri A Nusinow,Rebecca Bart,Titus Alicai

doi:10.3389/fpls.2017.01780

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.

Highlights

Development of superior crop varieties has relied on securing genetic gains through sexual recombination, induced random mutagenesis, and transgenic approaches
The nucleotide sequence of A. thaliana Phytoene desaturase (PDS) (AtPDS: NM117498) was used as bait to search for homologous sequences within the reference cassava genome in Phytozome 11 and a single copy of MePDS identified
The guide RNA (gRNA) were cloned into two separate binary vectors (MePDS-1 and MePDS-2) carrying the Cas9 gene (Figure 1B), Arabidopsis U6-26 promoter and gRNA scaffold (Figure 1C), and delivered by Agrobacterium into embryogenic cells of cassava

Summary

Introduction

Development of superior crop varieties has relied on securing genetic gains through sexual recombination, induced random mutagenesis, and transgenic approaches. Gene-editing technologies based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) make use of engineered nucleases to induce double strand breaks (DSB) at known DNA sequences within the genome. The CRISPR/Cas system depends on DNA or RNA sequence homology, in which targeting a specific DNA sequence requires only a 17–23-bp complementary nucleotide sequence called a guide RNA (gRNA). This versatility, plus high efficacy, and low cost have made CRISPR/Cas the most widely adopted of the three genome-editing technologies (Doudna and Charpentier, 2014). CRISPR/Cas9-induced mutations have been confirmed to be heritable across sexual generations (Brooks et al, 2014; Ding et al, 2016)

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Results

Conclusion