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.

Figure 1

Frequency of CRISPR/Cas9-induced mutation types. (A,B) Frequency of each mutation type for all of the mutations induced by the 10 constructs in the T0 generation. i: insertion; d: deletion; s: substitution; c: combined mutation. d#, number of base pairs (bp) deleted from the target site; i#, number of bp inserted at target site, c#, number of bp combined mutations. (C) Frequency of different mutation lengths regardless of the mutation types using the data from (A). (D) Percentage of different bases for the 1-bp insertion (i1 in (B)). (E) Detailed characterization of the different mutation types. Notes: i: insertion, d: deletion, s: substitution, c: combined mutation (more than one mutation type in one allele).

Figure 2

Genotyping of the BnaC5.DA1-sgRNA-L16 plants in the T0 and T1 generation. (A) Cropped gel image showing the PCR products derived from Cas9 in BnaC5.DA1-sgRNA-L16 in the T0 generation. (B) The genotype of BnaC5.DA1-sgRNA-L16 in the T0 generation. The PAM sequence is indicated with green. The sgRNA is indicated with red. Mutation sites are indicated with blue. (C) Cropped gel image showing the PCR products of Cas9 in different progeny from BnaC5.DA1-sgRNA-L16 in the T1 generation. +: pKSE401 was used as a positive control; −: gDNA of WT was used as a negative control. BnaC5.DA1-sgRNA-L1 (T0 and T1) was used as a positive control for the Cas9 insertion. The arrowheads indicate the position of the amplicons.

Figure 3

Phenotype and genotype of a BnaA6.RGA-sgRNA single mutant (T0) and a BnaRGA-sgRNA quadruple mutant (T1). (A) Morphology of BnaA6.RGA-sgRNA transgenic plants at the same age. L4 and L6: two individual T0 transgenic lines. CK: BnaA6.DA1-sgRNA (L10). Bar = 15 cm. (B) Genotype of BnaA6.RGA-sgRNA lines (L4 and L6) in the T0 generation. (C) Amino acid sequence alignment of the target sites derived from WT and two BnaA6.RGA-sgRNA transgenic lines (L4 and L6). The DELLA and TVHYNP domains are underlined. The amino acid deletion in the DELLA domain is caused by sgRNA1. The amino acid deletion in the TVHYNP domain is caused by sgRNA2. (D) Image showing the morphology of the quadruple mutant induced by BnaRGA-sgRNA (L46-T1) at the same age as the control. CK: WT plant. Bar = 15 cm. (E) Genotype of the quadruple mutant isolated from BnaRGA-sgRNA-L46 in the T1 generation. The PAM sequence is indicated with green. The sgRNA is indicated with red. The mutation sites are indictaed with blue. For the term n/m, m indicates the number of clones examined, and n indicates the number of clones showing the indicated genotype.