Production of Marker-free Mutants of Brassica campestris Mediated by CRISPR/Cas9 Through Vacuum Infiltration

doi:10.13560/j.cnki.biotech.bull.1985.2022-0042

Abstract

Abstract:

The objective is to acquire non-transgenic Brassica campestris mutants via CRISPR/Cas9 gene editing system，for achieving the application of CRISPR/Cas9 in B. campestris in planta transformation method and providing reference for application of marker-free gene editing technique. Using ‘49 Caixin’ as plant material and dehydrogenase gene（PDS）as target gene，total of 25 plants were transformed by in planta transformation via vacuum-infiltration method. The results showed that 3 of the 2 032 transformed seedlings were identified to have PDS gene editing，one of which was detected to have exogenous vector insertion and heterozygous editing of PDS gene，and the phenotype was consistent with that of the wild type. The other two seedlings had a dwarf，albino phenotype，and there was no insertion of foreign vector in their genomes. Different types of targeted mutagenesis of PDS gene were detected in these two seedlings compared with the wild type. In conclusion，gene editing can be achieved in B. campestris by CRISPR/Cas9 through in planta tissue culture-independent transformation，and particularly non-transgenic editing mutants without exogenous insertion can be obtained directly.

Key words: Brassica campestris, marker-free, vacuum infiltration transformation, tissue culture-independent, CRISPR/Cas9

ZONG Mei, HAN Shuo, GUO Ning, DUAN Meng-meng, LIU Fan, WANG Gui-xiang. Production of Marker-free Mutants of Brassica campestris Mediated by CRISPR/Cas9 Through Vacuum Infiltration[J]. Biotechnology Bulletin, 2022, 38(10): 159-163.

Figures/Tables 2

Table 1 Primers used in this study

引物名称 Primer name	序列 Sequence（5'-3'）	目的 Purpose
nCas9-IDF	CATACCTCCCAGAACACAAATAAGC	扩增nCas9片段
nCas9-IDR	ACTGAAGGGCAATAGTGAAGAATGT	扩增nCas9片段
gRNA-IDF	TGTCCCAGGATTAGAATGATTAGGC	扩增gRNA片段
RNA-IDR	CCCCAGAAATTGAACGCCGAAGAAC	扩增gRNA片段
PDS-1	ATGCAACTGATCAATGCGGT	扩增含有靶点的PDS基因片段
PDS-356	CGGGAATATCGCAGCTAAAG	扩增含有靶点的PDS基因片段
PDS-F	ATTGAACGAGAAGAAGCAAGCCT	载体构建
PDS-R	AAACAGGCTTGCTTCTTCTCGTT	载体构建

Fig. 1 CRISPR/Cas9-mediated gene editing and vacuum infiltration in planta transformation to creates PDS mutants in B. campestris （a）：DNA sequence of the first four exons（highlighted in different colors）of PDS of ‘49 Caixin’. The sgRNA at the end of the first exon is underlined and the PAM region CCC（complementary sequence is GGG）is shown in red letter.（b）：pBSE401 vector was used in vacuum infiltration transformation and the transcribed sgRNA was designed to have 19-base matches with the target. The vector carries Bar selection marker.（c）：Only plant #1 showed positive in Bar strip detection. And PDS gene sequencing of plant #1 showed sequence variation in the target region.（d）：Two of the 2 032 seedlings（plant #2 and plant #3）were weak albino，which was consistent with the phenotypes of PDS mutants. The picture shows plant #2.（e）：Sanger sequencing of plant #2 and plant #3 revealed the targeted mutagenesis of PDS gene in 2 plants compared with the wild type

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