CRISPR-Cas9 Targeted Mutagenesis Leads to Simultaneous Modification of Different Homoeologous Gene Copies in Polyploid Oilseed Rape (Brassica napus).
Janina BraatzHans-Joachim HarloffMartin MascherNils SteinAxel HimmelbachHans-Joachim HarloffPublished in: Plant physiology (2017)
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.
Keyphrases
- crispr cas
- wild type
- genome editing
- genome wide
- genome wide identification
- genome wide analysis
- circulating tumor
- copy number
- cancer therapy
- dna methylation
- cell free
- single molecule
- transcription factor
- aortic valve
- high fat diet
- gene expression
- dna damage
- single cell
- machine learning
- nucleic acid
- arabidopsis thaliana
- coronary artery disease
- big data
- amino acid
- heart failure
- genetic diversity
- transcatheter aortic valve replacement
- aortic stenosis