The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as effective strategy for generating site-specific mutations. Recently, CRISPR/Cas9-mediated genome editing system have been rapidly optimized and applied in crop genetic improvement. In this chapter, we present a simple and high-efficiency method for crating targeted gene mutation in allotetraploid cotton genome using CRISPR/Cas9 system. This chapter will employ GhMYB25-like A and GhMYB25-like D that derived from upland cotton (Gossypium hirsutum) A subgenome and the D subgenome, respectively, as an example to introduce the procedure of how to generate effective mutations in cotton genome using CRISPR/Cas9-based biotechnology. Based on our previous results, this CRISPR/Cas9 system can induce a proportion of 14.2-21.4% fragment truncation events in GhMYB25-like A and GhMYB25-like D genome sites. In addition, PCR product sequencing results suggest that the mutation frequencies that occurred in GhMYB25-like A and GhMYB25-like D DNA sites are 100% and 98.8%, respectively. More important, the off-target-caused mutation events have not been detected in our transgenic plants, even one of the putative off-target site only have one nucleotide mismatch with the designed GhMYB25 sgRNA. Thus, this CRISPR/Cas9 method might be an effective approach for targeted mutagenesis in cotton genome.
Keywords: CRISPR/Cas9; Cotton; Genome editing; Targeted mutagenesis.