DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration

Abstract

Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free CRISPR-Cas delivery system has potential to mitigate public concern about genetically modified organisms. Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. We generated mutants for genes involved in small interfering RNAs (siRNA) biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6) and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots. The ploidy level of these regenerants was not affected by PEG-Ca2+-mediated transfection, CRISPR reagents, or the target genes. By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. spsgs3 null T0 regenerants and sprdr6 null T1 progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type stock and pollination with wild-type pollen. The resulting seeds contained the mutated alleles. Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the wild type. Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum)

“…A DNA-free genome editing method requires efficient and reproducible shoot regeneration from single cells, which is still a challenge and can be highly genotype dependent (Peres et al 2001 ). A recent study reported protoplast regeneration via a DNA-free method on wild tomato, which is the closest study so far to cultivated tomato (Lin et al 2022 ). Based on a previously published protocol for cultivated tomato protoplast genome editing via RNP-based CRISPR/Cas9 (Nicolia et al 2021a ), we have improved the protoplast isolation process and solved the challenge of shoot regeneration from RNP-transfected protoplasts.…”

“…Although a high editing efficiency has been reported on tomato calli from transfected protoplasts of cultivated tomato, the shoot regeneration from RNP-transfected protoplasts is a bottleneck (Nicolia et al 2021a ). Very recently, (Lin et al 2022 ) reported the successful protoplast regeneration of wild tomato ( Solanum peruvianum ) harboring CRISRP/Cas9 mutations, with a mutation rate varying from 8.3% to 63.6%. However, for cultivated tomato, there are only a few old reports published on plant regeneration from unedited protoplasts (Morgan and Cocking 1982 ; Sakata et al 1987 ; Tan et al 1987 ).…”

Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum)

“…A DNA-free genome editing method requires e cient and reproducible shoot regeneration from single cells, which is still a challenge and can be highly genotype dependent (Peres et al 2001). A recent study reported protoplast regeneration via a DNA-free method on wild tomato, which is the closest study so far to cultivated tomato (Lin et al 2022).…”

“…Although a high editing e ciency has been reported on tomato calli from transfected protoplasts of cultivated tomato, the shoot regeneration from RNP-transfected protoplasts is a bottleneck (Nicolia et al 2021). Very recently (Lin et al 2022) reported the successful protoplast regeneration on wild tomato (Solanum peruvianum) harbouring CRISRP/Cas9 mutations, with a mutation rate varying from 8.3-63.6%. However, for cultivated tomato, there are only a few old reports published on plant regeneration from unedited protoplasts (Morgan and Cocking 1982 ;Sakata et al 1987;Tan et al 1987).…”

Enhancing HR Frequency for Precise Genome Editing in Plants

“…Protoplasts are a useful tool for implementing and monitoring HR-based genome editing ( Beard et al, 2021 ; Wright et al, 2005 ; Townsend et al, 2009 ; Puchta and Fauser, 2013 ; Zhu et al, 2020 ; Hsu et al, 2021 ). Working with protoplasts enables the convenient and efficient delivery of large quantities of donor template as well as DNA that encodes for sequence-specific nucleases ( Sant’Ana et al, 2020 ; Nicolia et al, 2021 ; Lin et al, 2022 ). It has been hypothesized that efficient delivery of large amounts of donor template results in increased HR-mediated GT efficiency ( Baltes et al, 2014 ).…”