CRISPR/Cas editing of a CPC gene in Arabidopsis thaliana

Abstract

BACKGROUND: Identification of target genes responsible for visible phenotypic effect may contribute to the development of transgene-free bioengineering strategies and application of crop varieties with edited genome. CAPRICE (CPC) is a single-repeat R3 MYB transcription factor, involved in anthocyanin biosynthesis and trichome formation. It is assumed that CPC controls the expression of Dihydroflavonol-4-reductase (DFR), a key gene of anthocyanin biosynthesis.

AIM: The aim of the study was to determine whether knockout of the CPC gene using CRISPR/Cas9 results in visible anthocyanin accumulation.

MATERIALS AND METHODS: Three guide RNAs were designed to excise a MYB domain from the CPC gene of Arabidopsis thaliana. Anthocyanin content and expression of CPC and DFR genes were studied in edited plants.

RESULTS: The expected 662 bp deletion was detected in 2,7% of glufosinate-resistant plants, however none of the mutations were homozygous. Four edited lines were studied in four generations. An upregulation of the DFR gene was observed in edited lines, however CPC gene expression, anthocyanin content and trichome development were not significantly different from those in control plants. Moreover, in A. thaliana pigmentation did not directly depend on DFR or CPC gene expression.

CONCLUSIONS: Our results suggest that CPC gene is involved in regulation of DFR gene expression and anthocyanin biosynthesis pathway, however in case of mutations plants might utilize other transcription factors to maintain homeostasis. Therefore, CPC gene is not a suitable target for CRISPR/Cas studies in Arabidopsis.

Keywords

DFR / anthocyanins / RT-PCR / genome editing

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Funding

Russian Science FoundationРоссийский научный фонд(20-74-10053)

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