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"

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."