Gene editing

CRISPR/Cas-based gene editing has revolutionized plant research and breeding. A major advantage of the technology is the ability to multiplex and target multiple genes simultaneously. In our research we are combining multiplex gene editing with doubled haploid breeding to rapidly generate homozygous edited lines. We are further developing technologies related to haploid induction, haploid identification and transgenerational gene editing. Finally, together with the Jacobs lab we are implementing novel CRISPR-based technologies such as base editing in maize.

 

Gene editing with DH breeding
Fig. 1: Combining multiplex gene editing and double haploid (DH) breeding in maize. Multiplex edited T0 plants are crossed with wild-type (WT) B104 plants resulting in BC1 plants that are either heterozygous or WT at each locus and can be selected for the absence of the EDITOR T-DNA to avoid further editing. BC1 plants are pollinated using a haploid inducer line carrying a GFP transgene (RWS-GFP). After pollination, embryos are isolated and haploid embryos are selected for the absence of GFP fluorescence (GFP-) and incubated on a medium containing colchicine. Colchicine-treated embryos are germinated in vitro, and chromosome doubling is confirmed by flow cytometry. Every haploid embryo and doubled haploid plant will show a random combination of edited loci. Doubled haploids (DH0) are self-pollinated to obtain genetically identical homozygous DH1 seeds. Adapted from Impens et al. (2023), New Phytol. 

 

GFP negative embryos
Fig. 2: GFP negative DH embryos
Gene edited DH1 seedlings
Fig. 3 Gene edited DH1 seedlings

 

 

 

 

 

 

 

 

 

 

Selected publications

  1. Impens L., Lorenzo C.D., Vandeputte W., Wytynck P., Debray K., Haeghebaert J., Herwegh D., Jacobs T.B., Ruttink T., Nelissen H., Inzé D. and Pauwels L.* (2023). Combining multiplex gene editing and doubled haploid technology in maize. New Phytol. 239, 1521-1532.
  2. Impens L., Jacobs T.B., Nelissen H., Inzé D. and Pauwels L.* (2022). Mini-review: Transgenerational CRISPR/Cas9 gene editing in plants. Frontiers in Genome Editing 4, 825042.
  3. Lorenzo C.D.°, Debray K.°, Herwegh D., Develtere W., Impens L., Schaumont D., Vandeputte W., Aesaert S., Coussens G., De Boe Y., Demuynck K., Van Hautegem T., Pauwels L., Jacobs T.B., Ruttink T., Nelissen H. and Inzé D.* (2023). BREEDIT: a multiplex genome editing strategy to improve complex quantitative traits in maize. Plant Cell 35, 218-238.