Targeted mutagenesis in the tetraploid switchgrass cultivar 'Alamo' using CRISPR-Cas9

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2016-01-01
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Merrick, Paul
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Shui-zhang Fei
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Horticulture
The Department of Horticulture was originally concerned with landscaping, garden management and marketing, and fruit production and marketing. Today, it focuses on fruit and vegetable production; landscape design and installation; and golf-course design and management.
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Abstract

Switchgrass (Panicum virgatum L.) is a highly self-incompatible perennial grass species which has been designated as a model bioenergy crop by the U.S. Department of Energy. To enhance breeding efforts of switchgrass we tested the efficacy of the targeted gene editing tool CRISPR-Cas9 in the tetraploid cultivar ‘Alamo’. Agrobacterium mediated transformation of embryogenic callus tissue averaged a transformation efficiency of 12.0% across experiments targeting four genes: Flowering Locus T (FT), APETALA 1 (AP1), Brassinosteroid Receptor Insensitive 1 (BRI1), and Phosphoglycerate Mutase (PGM). Out of 77 PCR confirmed transgenic events, a single event contained a heterozygous, CRISPR-Cas9 mediated 1 base pair deletion in the PGM target gene. Transgene silencing due to switchgrass’s highly methylated genome may play a role in the low mutation frequency. The mutation was obtained using a single sgRNA, however multiplex CRISPR using 2 sgRNA’s as well as tRNA constructs were also tested.

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Fri Jan 01 00:00:00 UTC 2016