CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus

Abstract

CRISPR/Cas9 is a valuable tool for both basic and applied research that has been widely applied to different plant species. Nonetheless, a systematical assessment of the efficiency of this method is not available for the allotetraploid Brassica napus—an important oilseed crop. In this study, we examined the mutation efficiency of the CRISPR/Cas9 method for 12 genes and also determined the pattern, specificity and heritability of these gene modifications in B. napus. The average mutation frequency for a single-gene targeted sgRNA in the T0 generation is 65.3%. For paralogous genes located in conserved regions that were targeted by sgRNAs, we observed mutation frequencies that ranged from 27.6% to 96.6%. Homozygotes were readily found in T0 plants. A total of 48.2% of the gene mutations, including homozygotes, bi-alleles, and heterozygotes were stably inherited as classic Mendelian alleles in the next generation (T1) without any new mutations or reversions. Moreover, no mutation was found in the putative off-target sites among the examined T0 plants. Collectively, our results demonstrate that CRISPR/Cas9 is an efficient tool for creating targeted genome modifications at multiple loci that are stable and inheritable in B. napus. These findings open many doors for biotechnological applications in oilseed crops.

Editing of an Alpha-Kafirin Gene Family Increases, Digestibility and Protein Quality in Sorghum

“…They achieved 100% editing by the delivery of the three, four, or five gRNA plasmid mixtures along with the Cas9 plasmid in Physcomitrella patens. Yang et al (2017) designed two gRNAs and successfully targeted three gene families (BnaRGA with four genes, BnaDA2 with two genes, and BnaFUL with three genes). Recently, Sánchez-León et al (2018) used two consensus gRNAs to target the wheat a-gliadin gene family with about 100 members and obtained one line containing 35 mutated genes.…”

The CRISPR/Cas revolution continues: From efficient gene editing for crop breeding to plant synthetic biology

“…Dwarf and semi‐dwarf plants have been generated via knockout of BolC.GA4a in rapeseed (Lawrenson et al ), ER1 , ER2 and SEC3a in rice (Ma et al ; Zhang et al ) and Dep1 in wheat (Zhang et al ). Semi‐dwarf rapeseed was also created by selecting mutants carrying deletions in the BaA6.RGA gene, whereby the translational reading frame is retained (Yang et al ). This principle provides the opportunity to achieve functional modifications rather than an entire loss‐of‐function of the target gene.…”

Development of pre-breeding diploid potato germplasm displaying wide phenotypic variations as induced by ethyl methane sulfonate mutagenesis

“…Cultivated potato is autotetraploid (2n = 4x = 48) with a narrow and highly heterozygous genetic base (Srivastava et al 2016). The genetic base of cultivated potato can be expanded by crossing with wild Solanum species, which are rich sources of disease and pest resistance along with yet untapped quality traits (Yang et al 2017a). However, interspecific and interploidy crosses between S. tuberosum and other Solanum species are proven to be difficult due to sexual incompatibility and difference in endosperm balance numbers (Watanabe 2015).…”