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

Journal Article / 2017

Braatz, Janina; Harloff, Hans-Joachim; Mascher, Martin; Stein, Nils; Himmelbach, Axel; Jung, Christian

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 ALCATRAZ (ALC) homoeologs. ALC is involved in valve-margin development and thus contributes to seed shattering from mature fruits. Knocking out ALC would increase shatter resistance to avoid seed loss during mechanical harvest. We obtained a transgenic T1 plant with four alc 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 non-chimeric double heterozygote. T-DNA and ALC 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.

Plant physiology

10.1104/pp.17.00426

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