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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (02): 227-235.doi: 10.3724/SP.J.1006.2018.00227

• Orginal Article • Previous Articles     Next Articles

Establishment of CRISPR/Cas9 Genome Editing System Based on GbU6 Promoters in Cotton (Gossypium barbadense L.)

Ji-Yang LI, Jian-Feng LEI, Pei-Hong DAI, Rui YAO, Yan-Ying QU, Quan-Jia CHEN, Yue LI, Xiao-Dong LIU*()   

  1. College of Agronomy, Xinjiang Agricultural University / Key Laboratory of Agricultural Biotechnology of Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China;
  • Received:2017-05-21 Accepted:2017-11-21 Online:2018-02-12 Published:2017-12-11
  • Contact: Xiao-Dong LIU E-mail:xiaodongliu75@aliyun.com
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31660433) and the Innovation Research Program for Graduate Students in Xinjiang (XJGRI2016055).

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Abstract:

CRISPR/Cas9 genome editing is a powerful tool for genes functional analyses, and the mutation of endogenous genes has been successfully implemented in many organisms using the tool. Two cloned U6 promoter from sea island cotton Xinhai 16 were used to construct CRISPR/Cas9 gene editing vectors with target (GbGGB and GBERA1) DNA fragments from Xinhai 16 respectively. Through PEG method, the core fragments (including GbU6::sgRNA and CAMV35S::Cas9) of the CRISPR/Cas9 gene editing vectors enriched by PCR method were transformed into the cotton protoplast prepared from the embryo callus of Xinhai 16. The mutation of endogenous target genes was successfully detected by a restriction enzyme PCR (RE-PCR) assay of protoplast genome. The cloning and sequencing of the PCR product, showed that the two Cas9-GbU6-sgRNA vectors could both induce targeted mutagenesis. Sequence analysis revealed that most of the mutations were nucleotide substitutions and the few were nucleotide deletion. The results indicate that the CRISPR/Cas9 gene editing vector system based on GbU6 promoter can realize targeted mutagenesis in sea island cotton, which provides an important technical basis for cotton functional genomics research.

Key words: cotton, protoplast, CRISPR/Cas9, genome editing

Fig. 1

Sketch map of genome editing structure of cotton (Gossypium barbadense L.) A: editing vector containing Cas9I; B: editing vector containing Cas9II."

Table 1

Primers used in this study"

引物名称
Primer name		 序列
Sequence (5'-3')		 引物名称
Primer name		 序列
Sequence (5'-3')
GbU6-4PERA1-sg1F GATTGTTCGCAGAATGCATGACGG GbU6-5PGGB-sg2F AAGTGCTCTGTCGAAGTACTGAAG
GbU6-5PERA1-sg1F AAGTGTTCGCAGAATGCATGACGG GbU6GGB-sg2R AAACCTTCAGTACTTCGACAGAGC
GbU6ERA1-sg1R AAACCCGTCATGCATTCTGCGAAC Test GGB-sgF AAGTGGAAAGAGAATGGCGAC
GbU6-4PERA1-sg2F GATTGTCTTTCGCAGAATGCATGA Test GGB-sgR AGCTAATTCGCTATTGCAATCAATC
GbU6-5PERA1-sg2F AAGTGTCTTTCGCAGAATGCATGA Test ERA1-sgF GTATCCTCTTACCGCCTTTC
GbU6ERA1-sg2R AAACTCATGCATTCTGCGAAAGAC Test ERA1-sgF CATACTTACTGAGATGGCTGT
GbU6-4PGGB-sg1F GATTGTGTCGAAGTACTGAAGCGG FJ-1F GTAAAACGACGGCCAG
GbU6-5PGGB-sg1F AAGTGTGTCGAAGTACTGAAGCGG FJ-1R CAGGAAACAGCTATGAC
GbU6GGB-sg1R AAACCCGCTTCAGTACTTCGACAC FJ-2F TAAACTGAAGGCGGGAAACG
GbU6-4PGGB-sg2F GATTGCTCTGTCGAAGTACTGAAG FJ-2F CGGTTCTGTCAGTTCCAAACG

Fig. 2

Restriction enzyme digestion identification of gene editing vector"

Fig. 3

Morphological changes of cotton protoplasts before and after transformation A: result of cotton protoplasts overnight microscopy test (×40); B: results of microscopic test overnight examination of cotton prtoplasts transformed into the core fragments of GbU6-5-GGB-sgRNA 1-Cas9 I vector (×40). Bar=100 μm."

Fig. 4

Schematic diagram of digestion /PCR for editing effect of CRISPR/Cas9 The red region is the enzyme-digested site that has been edited."

Fig. 5

Digestion /PCR for editing effect of CRISPR/Cas9 M: 2000 plus DNA marker. A: 1, 2: PCR amplification of GbU6-5P:: ERA1-sgRNA1-Cas9 I genome before and after enzyme digestion; 3, 4: PCR amplification of GbU6-5P:: GGB-sgRNA1-Cas9 I genome before and after enzyme digestion. B: 1, 2: PCR amplification of GbU6-5P:: ERA1-sgRNA2-Cas9 I genome before and after enzyme digestion; 3, 4: PCR amplification of GbU6-5P::GGB-sgRNA2-Cas9 I genome before and after enzyme digestion; CK1, CK2: PCR amplification of negative control genome before and after enzyme digestion."

Fig. 6

Sequencing of GbGGB-sgRNA2 target site that were edited (type of base substitution)"

Fig. 7

Sequencing peaks figure of GbGGB-sgRNA2 target site that were edited (type of base substitution) The red box indicates the region is base pair were changed; the black box indicates the PAM site area."

Fig. 8

Sequencing peaks figure of GbGGB-sgRNA2 target site that were edited (type of base deletion)The red box indicates the region of deletion sequence(B) compared with the control (A); C indicates the base deletion region."

Fig. 9

Mutation efficiency of GbGGB-sgRNA2 target site (guide RNA) and its two flanking region L30, L60, and L90 indicate the 30, 60, and 90 bp region on the left of guide DNA respectively; R30, R60, and R90 indicate the 30, 60, and 90 bp region on the right of guide DNA, respectively."

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