Simultaneous genetic transformation and genome editing of mixed lines in soybean (glycine max) and maize (zea mays)

This study was conducted by a cross-functional team of Bayer Crop Science R&D scientists from Plant Biotechnology and Global Breeding. Authors used a novel method, termed Transformation and Editing of Mixed Lines (TREDMIL) to demonstrate simultaneous transformation and editing of numerous elite genotypes in soybean and maize. In this method, many lines were mixed prior to the production of seed embryo explants and the mixed lines were subsequently transformed with genome editing machinery directing the creation of indels to the targeted genic regions through co-expression of soybean Dt1-specific or maize Bm3-specific CRISPR RNAs (crRNAs) and the editing nuclease Cas12a. After regeneration, line identity was deconvoluted via genotyping and the editing outcome was verified by amplicon sequencing.

Through genotyping of regenerated events, the authors first showed that 97% of 104 elite soybean genotypes (101) were simultaneously transformed and the recovered transformants were distributed across diverse maturity groups (MG) from 00 to VII. Similarly, 55% of 40 elite maize female inbreds (22) were simultaneously transformed and the transformants were distributed across diverse relative maturities (RM) from 92 to 117. Using amplicon sequencing, authors subsequently demonstrated that edits at Dt1 target sites were detected in 94% of soybean events representing all 101 transformed lines and that more than 80% transformed soybean lines produced edits in greater than 90% of the events produced by that line. Similarly, edits at Bm3 target sites were characterized in 69% of maize events and represented 17 out of 22 transformed female inbreds. These results clearly demonstrated highly efficient, genotype flexible transformation and genome editing with the seed embryo transformation system.

Authors further examined the editing outcome of TREDMIL experiments at crRNA target sites Dt1 and Bm3 to understand editing profiles across diverse germplasm. Across three Dt1 target sites, 1,506 events produced over 800 distinct edits and over 4,000 total edits. Across three Bm3 target sites, 96 events produced 95 distinct edits and over 200 total edits. The large spectrum of edits produced across germplasm could enable genomic discoveries at a much faster pace than analysis of those same edits in just a single germplasm. Furthermore, authors demonstrated that a distinct 7-bp deletion at the Dt-1389 site was detected in 45% (678 out of 1506) edited events across 98 different genotypes in soybean. Similarly, a distinct 3 bp deletion at the Bm3-2070 site was detected in 65% (11 out of 17) edited maize female inbreds. Creation of identical edits in different germplasm can provide a mechanism with which to assess germplasm-by-edit interactions earlier in a breeding program, which would enable more rapid decision making on the broad-spectrum efficacy of a given edit. These results illustrate that TREDMIL can help accelerate the development and deployment of customized crop varieties in the precision breeding future.

See the article:

https://link.springer.com/article/10.1007/s42994-024-00173-5