Co-transfection Strategy for Efficient Cas-free Gene Editing in T0 Generation Plants

Co-transfection Strategy for Efficient Cas-free Gene Editing in T0 Generation Plants

Transgene-free genome editing of T0 generation plants is highly desirable but challenging, especially in perennials and vegetatively propagated plants. Ribonucleoprotein complex (RNP) were successfully used to produce transgenic-free plants, which usually required transformation of protoplasts. However, plant protoplast regeneration remains technically challenging and/or restricted to specific plant species/genotypes.

In addition to the RNP approach, there is also a strategy to produce transgenic-free plants directly from Agrobacterium. Although genome editing by Agrobacterium produces transgenic plants, most T-DNAs carrying Cas/gRNA will not be integrated into the host chromosome but will be present in the nucleus where they will be transcribed, leading to transient expression of the carried genes. However, the main drawback of using this approach to produce transgene-free plants is that most transformants are wild-type, most edited plants are mosaic/chimeric or heterozygous, and additional generations are required to identify transgene-free and homozygous/biallelic mutants. In addition, previous genome editing by transient expression of Cas/gRNA constructs is usually performed in the absence of selection pressure, making it difficult, laborious, and time-consuming to distinguish edited from unedited plants.

On March 2, 2023, bioRxiv published online the latest research results of Nian Wang's team at the Citrus Research and Education Center at the University of Florida: "Efficient transgene-free genome editing in plants in the T0 generation based on a co-editing strategy". Based on the production of transgene-free plants based on Agrobacterium infestation, they developed a co-editing strategy to fusion transiently express cytosine base editor (CBE)/gRNA-Cas12a/crRNA-GFP for the production of transgene-free, gene-edited plants.

Specifically, base editing of the ALS gene using CBE/gRNA confers a herbicide-resistant phenotype as a selectable marker without negatively affecting the plant phenotype. Cas12a/crRNA is used to edit the gene of interest. GFP was used to select transgene-free transformants. Using this approach, various genes (single or multiple) in T0-generation tomato, tobacco, potato and citrus were efficiently generated in transgene-free genome edited plants. The biallelic/homozygous transgene-free mutation rates of the target gene in the herbicide-resistant transformants ranged from 8% to 50%.

Validation of the Co-editing Strategy in Tomatoes

They first investigated whether transgene-free gene-edited tomatoes could be obtained in the T0 generation by base-editing SlALS1 alone. The CBE-Cas12a-GFP-SIALS1 construct was constructed to edit the SIALS1 gene using CBE to target the proline residue at position 186 (Pro186). More than 20 chlorsulfuron-resistant lines without green fluorescence were obtained, which were presumed to be transgenic-free genome editing lines. No GFP gene was detected in 5 GFP-negative lines randomly selected, and the editing results were further confirmed by Sanger sequencing, and no off-target editing occurred, and no transgene was confirmed by whole genome sequencing. These results suggest that transgene-free gene-edited plants can be obtained in the first generation by base editing the ALS gene and selecting for chlorsulfuron-resistant and GFP-negative plants.

They next tested the co-editing strategy by CBE-editing ALS and Cas12a-editing SlALS1 in tomato using the CBE-Cas12a-GFP construct. A total of 12 herbicide-resistant transformants without green fluorescence were selected for genotyping. In 12 herbicide-resistant transformants, the SIALS1 gene was edited in all lines. Phenotypes of biallelic SlER mutants include compact structures, short petioles, dense clusters, and enlarged SAMs. To test the heritability of the mutation, seeds of sler-4 T0 plants were germinated and the resulting seedlings were genotyped. Genotyping analysis found that mutations in the SlER gene were indeed heritable, with offspring either homozygous (inheriting one of the two edited alleles) or biallelic (identical to their parents).

Transgene-free Genome Editing of T0 Generation Tomato

They next investigated whether GM-free multiple genome editing in tomato could be achieved in the first generation. SlEDS1 and SlPAD4 were co-edited with SlALS1. Among the 18 GFP-negative transformants, 6 lines contained biallelic/homozygous editing of SlEDS1 and SlPAD4, and 2 lines were biallelic edited only in SlEDS1 but not in SlPAD4. Based on GFP observation and PCR analysis of the GFP and Cas12a genes, the edited lines were transgene-free.

Likewise, multiple gene editing was performed on SlDMR6 and SlINVINH1 with SlALS1. Three biallelic/homozygous SlDMR6/SlINVINH1 double mutants were obtained from 17 GFP-negative transformants. Four homozygous/biallelic slinvinh1 single mutants were also obtained. These edited lines are transgene-free based on GFP observation and PCR analysis of GFP and Cas12a genes.

In summary, using this strategy can effectively achieve transgene-free and multiple gene editing of the T0 generation tomato.

Expand The Scope of Verification

Subsequently, they verified whether the T0 generation could produce transgene-free genome editing in tobacco, potato, and citrus.

In tobacco, NtALS and NtPDS are co-edited. As expected, chlorsulfuron-resistant tobacco plants contained the NtALS mutation, whereas wild-type N. tabacum did not.

Co-editing of the StDMR6 and StALS genes in potato with a single crRNA targeting a conserved region in the first exon of the four StDMR6 alleles. Among the 15 GFP-negative transformants, 10 wild-type and 5 heterozygous mutants were obtained. These results suggest that this strategy can generate transgene-free, gene-edited potatoes in the T0 generation, but the generation of tetra-allelic mutants requires further optimization.

Co-edited LOB1 and CsALS genes in citrus, 107 pummelo shoots were obtained after screening for resistance to chlorsulfuron, of which 4 were GFP-positive and 103 were GFP-negative. After screening, 4 Cas-Free lines were finally obtained, and 4 transgene-free genome-edited lines contained homozygous/biallelic mutations.

In this study, the team developed an efficient genome editing toolkit to co-edit the ALS gene and the gene of interest through Agrobacterium-mediated transient expression to generate transgenic genome-edited plants in the T0 generation. It has been successfully used for genome editing in tomato and tobacco (annual plants), citrus (perennial tree crops) and potato (a clonal tetraploid crop).

Reference:

Huang, X., et al. Efficient transgene-free genome editing in plants in the T0 generation based on a co-editing strategy. bioRxiv. (2023). https://doi.org/10.1101/2023.03.02.530790