CRISPR-Cas9 Targeted Mutagenesis Leads to Simultaneous Modification of Different Homoeologous Gene Copies in Polyploid Oilseed Rape (Brassica napus)
CRISPR-Cas9 Targeted Mutagenesis Leads to Simultaneous Modification of Different Homoeologous Gene Copies in Polyploid Oilseed Rape (Brassica napus)
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DOI：
10.1104/pp.17.00426
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发表时间：
2017-06-01
期刊：
影响因子：
7.4
通讯作者：
Jung, Christian
中科院分区：
文献类型：
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作者：
Braatz, Janina;Harloff, Hans-Joachim;Jung, Christian
在多倍体物种中，由于基因冗余，通过随机突变来改变一个性状是非常低效的。我们用针对两个恶魔岛(ALC)同源基因的CRISPR-Cas9构建载体稳定地转化了四倍体油菜(Brassica Napus)。ALC参与了气门边缘的发育，因此有助于成熟果实的种子破碎。敲除ALC将增加抗破碎性，以避免在机械收获时种子损失。我们利用单个靶标序列获得了含有4个alc突变等位基因的转基因T1植株。所有突变均稳定遗传给T2代。T2代没有任何野生型等位基因，证明潜在的T1是非嵌合型双杂合子。T-DNA和ALC基因座没有连锁，表现为T2代的随机分离。因此，我们可以选择在第一代中已经缺乏T-DNA的双突变体。然而，全基因组测序数据显示，至少有五个独立的载体主干序列插入。我们没有在与目标序列同源的两个基因组区域中检测到任何脱靶效应。通过CRISPR-Cas9突变同时改变多个同源基因而不产生任何背景突变，将为利用突变基因在油菜育种中提供新的机会。
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.