Biology subjects

Gordon-Kamm, W.

Publications and source records attributed to Gordon-Kamm, W..

3 recordsLinked to original sources

bioRxiv2021

Wuschel2 enables highly efficient CRISPR/Cas-targeted genome editing during rapid de novo shoot regeneration in sorghum

For many important crops, including sorghum, the use of CRISPR/Cas technology is limited not only by the delivery of the gene-modification components into a plant cell, but more critically, by the ability to regenerate a fertile plant from the engineered cell through conventional tissue culture methods. Here, we report that Wuschel2 (Wus2)-enabled sorghum (Sorghum bicolor L.) transformation increases not only the efficiency of transgenic plant recovery, but also increases the observed efficiency of CRISPR/Cas-targeted genome editing within the population of T0 plants. Using Agrobacterium-mediated transformation, we have demonstrated Wus2-induced direct somatic embryo formation and regeneration, bypassing genotype-dependent callus formation and significantly shortening the tissue culture cycle time. This method also increased regeneration capacity, resulting in higher transformation efficiency across different sorghum genotypes. Subsequently, advanced excision systems and "altruistic" transformation technology have been developed to generate high-quality morphogenic gene-free and/or selectable marker-free sorghum events. Finally, we demonstrated up to an additional 6.8-fold increase in CRISPR/Cas9-mediated gene dropout frequency using Wus2-enabled, Agrobacterium-mediated transformation (compared to the control treatment with no Wus2), across various targeted loci in different sorghum genotypes. Wus2-enabled genome editing may be applicable to other crops and increasingly popular in planta transformation strategies.

developmental biology↗

bioRxiv2020

An efficient gene excision system in maize

Use of the morphogenic genes Baby Boom (Bbm) and Wuschel2 (Wus2), along with new ternary constructs, has increased the genotype range and the type of explants that can be used for maize transformation. In addition, altering the ectopic expression pattern for Bbm/Wus2 has resulted in rapid maize transformation methods that are faster and applicable to a broader range of inbreds. However, expression of Bbm/Wus2 can compromise the quality of regenerated plants, leading to sterility. We reasoned excising morphogenic genes after transformation but before regeneration would increase production of fertile T0 plants. We developed a method that uses an inducible site-specific recombinase (Cre) to excise morphogenic genes. The use of developmentally regulated promoters, such as Ole, Glb1, End2 and Ltp2, to drive Cre enabled excision of morphogenic genes in early embryo development and produced excised events at a rate of 25%-100%. A different strategy utilizing an excision-activated selectable marker produced excised events at a rate of 53.3%-68.4%; however, the transformation frequency was lower (12.9%-49.9%). The use of inducible heat shock promoters (e.g. Hsp17.7, Hsp26) to express Cre, along with improvements in tissue culture conditions and construct design, resulted in high frequencies of T0 transformation (29%-69%), excision (50%-97%), usable quality events (3.6%-14%), and few escapes (non-transgenic; 14%-17%) in three elite maize inbreds. Transgenic events produced by this method are free of morphogenic and marker genes.

plant biology↗

bioRxiv2020

Efficient Gene Targeting in Maize using Inducible CRISPR-Cas9 and Marker-Free Donor Template

CRISPR-Cas9 is a powerful tool for generating targeted mutations and genomic deletions. However, precise gene insertion or sequence replacement remains a major hurdle before application of CRISPR-Cas9 technology is fully realized in plant breeding. Here we report high frequency, selectable marker-free intra-genomic gene targeting (GT) in maize. Heat shock-inducible Cas9 was used for generating targeted double-strand breaks (DSBs) and simultaneous mobilization of the donor template from pre-integrated T-DNA. The construct was designed such that release of the donor template and subsequent DNA repair activated expression of the selectable marker gene within the donor locus. This approach generated up to 4.7% targeted insertion of the donor sequence into the target locus in T0 plants, with up to 86% detected donor template release and 99% mutation rate were observed at the donor loci and the genomic target site, respectively. Unlike previous in planta or intra-genomic homologous recombination reports, that required multiple generations and extensive screening, our method provides non-chimeric, heritable GT in the T0 generation.

plant biology↗