CRISPR-Cas9 Targeted Mutagenesis Leads to Simultaneous Modification of Different Homoeologous Gene Copies in Polyploid Oilseed Rape ( Brassica napus )

In polyploid species, altering a trait by random mutagenesis is highly inefficient due to gene redundancy. We have stably transformed tetraploid oilseed rape ( ) with a CRISPR-Cas9 construct targeting two ( ) homoeologs. is involved in valve margin development and, thus, contributes to seed shatteri...

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Published in:Plant physiology (Bethesda) Vol. 174; no. 2; p. 935
Main Authors: Braatz, Janina, Harloff, Hans-Joachim, Mascher, Martin, Stein, Nils, Himmelbach, Axel, Jung, Christian
Format: Journal Article
Language:English
Published: United States 01.06.2017
Subjects:
Base Sequence
Brassica napus - genetics
Brassica napus - growth & development
CRISPR-Cas Systems - genetics
DNA, Bacterial - genetics
Gene Dosage
Gene Targeting
Genome, Plant
Inheritance Patterns - genetics
Mutagenesis, Insertional - genetics
Mutation - genetics
Polyploidy
Seeds - genetics
Sequence Homology, Nucleic Acid
Transformation, Genetic
ISSN:1532-2548, 1532-2548
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Summary:In polyploid species, altering a trait by random mutagenesis is highly inefficient due to gene redundancy. We have stably transformed tetraploid oilseed rape ( ) with a CRISPR-Cas9 construct targeting two ( ) homoeologs. is involved in valve margin development and, thus, contributes to seed shattering from mature fruits. Knocking out would increase shatter resistance to avoid seed loss during mechanical harvest. We obtained a transgenic T1 plant with four mutant alleles by the use of a single target sequence. All mutations were stably inherited to the T2 progeny. The T2 generation was devoid of any wild-type alleles, proving that the underlying T1 was a nonchimeric double heterozygote. T-DNA and loci were not linked, as indicated by random segregation in the T2 generation. Hence, we could select double mutants lacking the T-DNA already in the first offspring generation. However, whole-genome sequencing data revealed at least five independent insertions of vector backbone sequences. We did not detect any off-target effects in two genome regions homologous to the target sequence. The simultaneous alteration of multiple homoeologs by CRISPR-Cas9 mutagenesis without any background mutations will offer new opportunities for using mutant genotypes in rapeseed breeding.
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ISSN:1532-2548
1532-2548
DOI:10.1104/pp.17.00426