Targeted genome editing in polyploids: lessons from Brassica

Abstract

CRISPR-mediated genome editing has emerged as a powerful tool for creating targeted mutations in the genome for various applications, including studying gene functions, engineering resilience against biotic and abiotic stresses, and increasing yield and quality. However, its utilization is limited to model crops for which well-annotated genome sequences are available. Many crops of dietary and economic importance, such as wheat, cotton, rapeseed-mustard, and potato, are polyploids with complex genomes. Therefore, progress in these crops has been hampered due to genome complexity. Excellent work has been conducted on some species of Brassica for its improvement through genome editing. Although excellent work has been conducted on some species of Brassica for genome improvement through editing, work on polyploid crops, including U's triangle species, holds numerous implications for improving other polyploid crops. In this review, we summarize key examples from genome editing work done on Brassica and discuss important considerations for deploying CRISPR-mediated genome editing more efficiently in other polyploid crops for improvement.

Keywords: Brassica; CRISPR; crop improvement; genome editing; polyploid crops.

Figure 1

Schematic illustration of the steps involved in the development of transgene-free genome-edited Brassica plants. (A) shows the identification and retrieval of target gene sequences from public databases, such as GenBank or The Brassicaceae Database (BRAD V3.0; http://brassicadb.cn). The sequences are aligned to identify potential domains for designing gRNA. (B) shows the gRNA designing, construction of plant expression vectors followed by transformation of that construct into Agrobacterium strain for plant transformation. (C) represents Agrobacterium¬- mediated Brassica transformation using cotyledonary explants, shoot regeneration on the selection medium, root induction, establishment of the plants in compost, and screening of the putative transformants using techniques such as T7 endonuclease I assay, Sanger sequencing, or qPCR. (D) illustrates the process of eliminating marker genes to obtain transgene-free edited plants through Mendelian segregation. Transgene-free edited plants can also be obtained by programmed self-elimination, using suicidal genes, and can be immediately identified using fluorescent genes. Another way of identification of transgene-free edited plants is by incorporating genes that confer distinct phenotypes, such as early flowering (Bolting assisted selection) or fluorescence. Some parts of the picture were created using BioRender.com.