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

• Published in: Scientific reports Vol. 7; no. 1; pp. 7489 - 13
• Main Authors: Yang, Hong, Wu, Jia-Jing, Tang, Ting, Liu, Ke-De, Dai, Cheng
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 08.08.2017; Nature Publishing Group; Nature Portfolio
• Subjects: 631/449/447/2311; 631/61/447; Agrobacterium - genetics; Alleles; Amino Acid Sequence; Base Sequence; Biotechnology; Brassica napus; Brassica napus - genetics; Brassica napus - metabolism; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems; Gene Editing - methods; Genes; Genes, Plant; Genetic Loci; Genome editing; Genomes; Genotype; Heritability; Heterozygotes; Homozygote; Homozygotes; Humanities and Social Sciences; multidisciplinary; Mutation; Mutation Rate; Oilseed crops; Oilseeds; Phenotype; Plant species; Rape plants; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Science; Science (multidisciplinary); Sequence Alignment; Tetraploidy
• DOI: 10.1038/s41598-017-07871-9
• ISSN: 2045-2322

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.