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

Abstract

In polyploid species, altering a trait by random mutagenesis is highly inefficient due to gene redundancy. We have stably transformed tetraploid oilseed rape (Brassica napus) 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 nonchimeric 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.

Figure 1.

Nonhomologous end joining-mediated knockout of two BnALC homoeologs. A, CRISPR-Cas9 target upstream of the bHLH domain of BnALC. The protospacer-adjacent motif (PAM) is underlined. The boxed target sequence was based on BnaA.ALC.a and comprised a single-nucleotide polymorphism (SNP) to BnaC.ALC.a at position 10, highlighted in red. B, Four CRISPR-Cas9-induced mutant alleles detected by Sanger sequencing of a single double heterozygous T1 rapeseed plant. The size of each deletion/insertion and the name of the allele are indicated at right. The inserted base of allele C₂ is highlighted by the red box.

Figure 2.

Alignment of the CRISPR-Cas9 target sequences from BnaA.ALC.a and BnaC.ALC.a in comparison with two potential off-target sites identified in the reference genome of Darmor-bzh by a BLAST search. The PAMs are underlined. SNPs are highlighted in red.

Figure 3.

Growth types of CRISPR-Cas9 alc mutants resemble the wild type, while siliques are more shatter resistant. A, T1 and T2 rapeseed plants carrying CRISPR-Cas9-mutated BnALC alleles next to cv Haydn, which had been used for transformation. Plants were grown in 11- × 11-cm pots in the greenhouse. B, alc mutations show no visible effect on mature siliques of T1 and T2 mutants compared with cv Haydn. C, Results of shatter resistance measurements of alc T2 in comparison with cv Haydn. Peak tensile forces are displayed as means of siliques grouped according to their length. Error bars represent sd. A significant difference is indicated by the asterisk (Student’s t test, P < 0.05).