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

Abstract 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 T0 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.