Targeting PDS gene to establish a transgene-free genome editing system in sorghum

Genome editing in plants using CRISPR/ Cas9 typically involves integrating transgenic constructs into plant genome. However, a challenge arises after the target gene is successfully edited, transgene elements such as Cas9 , gRNA cassette, and selective marker genes remain integrated. This integration of transgenes causes regulatory and environmental concerns, particularly for commercialization. In addressing this issue, we present the establishment of a transgene-free genome editing system in sorghum, achieved through transient gene expression without selection. We selected the phytoene desaturase ( PDS ) gene as the target due to its capacity to induce a visible phenotypic change, namely albinism, upon mutation. Following microprojectile co-transformation with maize optimised Cas9 vector and a gRNA cassette with kanamycin resistance gene, immature embryo (IE) derived tissues were divided into two groups (selection and non-selection) and deployed as parallel experiments. Remarkably, 4 out of 18 homozygous/biallelic editing lines in the non-selection group were identified as transgene-free lines in the T ₀ generation, with no traceable transgenes. Conversely, no transgene-free editing line was achieved in the selection group. This strategy not only enables to regenerate transgene-free genome-edited lines more efficiently but also saves one generation of time by eliminating the need for self-crossing or out-crossing. Our results displayed the feasibility of achieving transgene-free genome-edited plants within a single generation in sorghum. Furthermore, this approach opens avenues for vegetatively propagated crops like pineapple, sugarcane, and banana to obtain transgene-free genome-edited lines, facilitating their commercialization.