Multiplex CRISPR/Cas9-mediated genome editing to address drought tolerance in wheat

Abstract

Genome editing tools have rapidly been adopted by plant scientists for crop improvement. Genome editing using a multiplex sgRNA-CRISPR/Cas9 genome editing system is a useful technique for crop improvement in monocot species. In this study, we utilized precise gene editing techniques to generate wheat 3'(2'), 5'-bisphosphate nucleotidase (TaSal1) mutants using a multiplex sgRNA-CRISPR/Cas9 genome editing system. Five active TaSal1 homologous genes were found in the genome of Giza168 in addition to another apparently inactive gene on chromosome 4A. Three gRNAs were designed and used to target exons 4, 5 and 7 of the five wheat TaSal1 genes. Among the 120 Giza168 transgenic plants, 41 lines exhibited mutations and produced heritable TaSal1 mutations in the M₁ progeny and 5 lines were full 5 gene knock-outs. These mutant plants exhibit a rolled-leaf phenotype in young leaves and bended stems, but there were no significant changes in the internode length and width, leaf morphology, and stem shape. Anatomical and scanning electron microscope studies of the young leaves of mutated TaSal1 lines showed closed stomata, increased stomata width and increase in the size of the bulliform cells. Sal1 mutant seedlings germinated and grew better on media containing polyethylene glycol than wildtype seedlings. Our results indicate that the application of the multiplex sgRNA-CRISPR/Cas9 genome editing is efficient tool for mutating more multiple TaSal1 loci in hexaploid wheat.

Keywords: CRISPR-Cas9; Sal1 gene; drought tolerance; stomata; wheat.

Figure 1.

A diagram of the six TaSal1 genes present in Giza-168 hexaploid wheat showing the exons regions (blue boxes) and the 5’ (Orange arrow) middle (yellow arrow) and 3’ (blue arrow) locations of the gRNA. The * in TaSal1^4A- represents the location of the extra C inserted into the coding region downstream of the middle target sequence, * in TaSal1^7D represents single nucleotide mismatch (C instead of T) at the sgRNA.

Figure 2.

Giza-168 wheat transformation and regeneration stage using immature embryos. A & B: isolated embryos; C, D, E & F: callus formation on callus induction media; G and H: shoot formation on shoot induction media; I and J: shoot elongation and multiplication; K: root formation on root induction media; L and M: acclimatization of putative transgenic plant and N: putative transgenic plant maturation and seed formation.

Figure 3.

Stages of maturity of well-watered TaSal1 mutant Giza 168 A: Seeds obtained from transgenic plants (T₀), B: Status of the plants 37 days post germination, C: 60 days post germination, D: Wheat plants developing spikes harvested from wildtype Giza168 and GEL2 mutant plants grown in the same well-watered greenhouse.

Figure 4.

(a) Leaf of TaSal1 M₂ mutated lines with different rolled shapes. (b) Stems of TaSal1 M₂ mutated plants showing bending shape compared to the control.

Figure 5.

Transverse sections of leaves in control and M₂ of Giza 168 cultivar. A: Control from non-transgenic plant showing open stomata B: mutated line GEL119 showing closed stomata (blue arrows). Large bulliform cells in leaf blade of the mutated line compared to the control (black arrows). Intensive sclerification around the vascular bundles in leaf bade of GEL119 (red arrows).

Figure 6.

Scan electron microscopy scanning young leaves for Giza168 M₂ lines stomata and control (non-mutated plant) from well-watered plants.

Figure 7.

The length and the width of stomata guard cells of the 5 TaSal1 mutated lines compared to the control from well-watered plants.

Figure 8.

The seed germination on MS media supplemented with PEG. A: wild-type Giza168 seeds germinated on different concentration of PEG; B: control, E-02 and E-35 on MS media supplemented with 20% PEG.