High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system

Abstract

Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene-specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site-specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2-edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7-100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode-based high-throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1-edited T0 plants and it matched well with Sanger sequencing results. No off-target editing was detected at the potential off-target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.

Keywords: CRISPR/Cas9; allotetraploid; cotton; genome editing; high-throughput sequencing.

Figure 1

DsRed2 mutation is induced by the CRISPR/Cas9 system. (a) Seeds of wild‐type cotton YZ1 (upper row) and a DsRed2 overexpression line (bottom row). (b) and (c) Regenerated somatic embryos of the control line and two mutants (mR1 and mR2) in the white light field (b) and a red fluorescence field at an excitation wavelength of 530 to 550 nm (c). (d) to (o) Leaves and young seedlings from corresponding plants in (b) were observed in the white light field (d, e, f, j, k, l) and the red fluorescence field (g, h, i, m, n, o). Bar in (a) is 5 mm, in (b) to (o) is 2 mm. Sanger sequencing of somatic embryos (p) and two independent mutants (q) at the DsRed2 target sites are exhibited. The sgRNA target sites are highlighted in green background. PAM regions are highlighted in orange. Nucleotide deletions or insertions are shown in red, with details labelled at right. The gaps between the paired sgRNAs are in dotted line, and their lengths are labelled above. WT, the wild type. m, mutation clones.

Figure 2

Genome editing of GhCLA1 generates albino cotton plants. Regenerated somatic embryos of the control line (a) and five mutants (b–f) are shown. A green seedling among the albino plantlets is pointed out with a red arrow (f). Young plantlets of the corresponding control line (g), four mutants (h–k) and mosaic and green ‘mutants’ (l) are exhibited. Green part of the mosaic plant is highlighted in a magnified dash box and a red arrow (l). Bars in (g) to (l) are 1 cm.

Figure 3

Sanger sequencing of somatic embryos at GhCLA1 target sites. (a) Genome editing at sgRNA7 and sgRNA8 sites. (b) Genome editing at sgRNA9 and sgRNA10 sites. The sgRNA target sites and the PAM regions are highlighted in green and orange background, respectively. Nucleotide deletions or insertions are shown in red, with details labelled at right. The gaps between the omitted nucleotides are in dotted line, and their lengths are labelled above. A 20 bp insertion is shown in brackets under the sequence. WT, the wild type. m, mutated clones.

Figure 4

Mutation at GhCLA1 target sites with T7E1 digestion assay and Sanger sequencing. (a) GhCLA1 and Cas9 positivity check of the wild type (WT), control (CK) and mutants. Mutants m3 and m9 are in chimeric and green phenotype. Mutants m6, m4, m3 and m9 were targeted at sgRNA7 and sgRNA8 sites. Mutants m24 and m26 were targeted at sgRNA9 and sgRNA10 sites. (b) and (c) T7E1 digestion assay of the mutants at the target sites. M, marker. A large fragment deletion was occurred in a representative albino mutant m4. Its Sanger sequencing results (d) and the detailed chromatograms (e) at the target sites are illustrated. In (d), the SNP is highlighted in black. In (e), the top chart illustrates the large fragment deletion of one GhCLA1 copy. The charts at middle and bottom represent mutations at separate target sites of the same gene locus. The PAM regions and mutated target sites are underlined with black and red lines, respectively.

Figure 5

DsRed2 mutation is genetically inheritable from T0 to T1 generation. Seeds (a) and seedlings (b) of YZ1, the control and three representative T1 plants. (c) PCR analysis of Cas9 and DsRed2 in YZ1, CK, the T0 mutant mR1 and T1 progenies. (d) Genotyping of the target DsRed2 gene of independent T0 plants and their T1 progenies. Results for the T0 plants are highlighted in blue textbox. Mutated sites are highlighted in red with grey background. Bars in (a) and (b) are 2 and 3 cm, respectively.

Figure 6

Barcode‐based high‐throughput sequencing is applicable to detect a large population of mutants in a run. (a) Pipeline of the barcode‐based sequencing. The barcodes (coloured) are added to 5′ end of the primer. (b) A representative alignment of sequencing reads from GhCLA1‐edited mutant m27. The sgRNA target sites in the reference DNA are highlighted in green. The barcodes in sequencing reads are highlighted in orange and yellow. The regions in blue box represent the sequencing gap for the paired reads. The SNP against the sgRNA9 site is illustrated in red. Nucleotide deletions at the target sites are in blank, with details labelled at right. The brown and purple lines represent the origination of the reads from D and A subgenome, respectively.