Multi-Site Genome Editing in Cotton using CRISPR-Cas9 System

Cotton (Gossypium hirsutum) is an allotetraploid plant with a complex genome, with most genes having multiple copies. Due to its high efficiency, the CRISPR-Cas9 system was used by the team of Pengcheng Wang from Huazhong Agricultural University to generate two sgRNAs in a single vector and conduct multiple-site genome editing in cotton.

The team targeted the exogenously transformed gene, Discosoma red fluorescent protein2 (DsRed2) in DsRed2-overexpressing cotton, which has a red seed phenotype. Meanwhile, the endogenous gene GhCLA1, responsible for chloroplast development, was also chosen as a target of CRISPR-Cas9.

The T0 generation of the DsRed2-edited plants had traits similar to wild types, with no red fluorescence in their seeds. This trait was also inherited by 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 ranged from 66.7–100%. Furthermore, no off-target mutations were detected.

These results prove that CRISPR-Cas9 system is a reliable approach for allotetraploid cotton genome editing.

For more on this study, read the article in Plant Biotechnology Journal.