TABLE 1.

Applications of genome editing that may or may not be possible to achieve using traditional breeding methods. The level of technical difficulty and/or time needed to for different types of genome editing to be replicated using traditional breeding methods are indicated by: ✓✓✓ relatively facile; ✓✓ possible in longer time frames; ✓ technically challenging, laborious or needing much long time frames; × not possible in any reasonable timeframe.

Category of genome editing	Example of genome editing with references		Ease of replication via traditional breeding methods	Justification	Example references
SDN-0 Non-enzymatically active CRISPR molecules (dCas9) used to direct DNA methylases or acetyltransferases to alter the epigenetic status of targeted genomic locations with no change in the genome sequence.	Altered DNA methylation	Séré and Martin, (2020), Ghoshal et al. (2021)	Although types and locations of epigenetic marks in a plant can vary over space and time, natural epigenetic variation is more frequent than genetic mutations and specific epigenetic status could, in theory, be selected for	Natural epigenetic variation is widespread, heritable and contributes to plant adaptation. Intentionally or not, it will have been selected for (or against) in traditional breeding.	Becker et al. (2011); Schmid et al. (2018); Varotto et al. (2020)
			Epigenetic alteration at single locus ✓✓
	Altered Histone acetyltransferase activity	Roca Paixão et al. (2019)	Epigenetic alteration at multiple loci in same plant ✓
SDN-1/ SDN-2 Site-directed nuclease with or without repair template can readily generate SNPs and INDELs in a specific diploid or polyploid parental genotype. These may be homozygous in the first generation.	Gene knockout / loss of function alleles via premature stop codon, frameshift etc.	Chandrasekaran et al. (2016), Bull et al. (2018)	Loss of function/ SNP / INDEL at a single pre-determined genomic location ✓✓✓	Insertions, deletions, inversions, and duplications of DNA sequences occur throughout the genome. Forward and reverse genetic screening for individuals possessing equivalent mutations in some crops is facile. Traditional methods to combine multiple mutations at different loci using marker assisted selection is also possible in some crop species but more challenging or impossible in vegetatively propagated, perennial, self-incompatible etc crops. The generation of de novo, functional gene sequences via iterative generation/selection of multiple, independent, contiguous mutations, is effectively impossible using current traditional breeding approaches. However, the introgression of one or multiple genes from a crossable species is relatively facile (see below).	Funatsuki et al. (2014); Hasan et al. (2021)
			Loss of function/ SNPs / INDELs at multiple, independent pre-determined genomic locations in same plant ✓✓
	Gene knockout / loss of function alleles via multiple SDN excision.	Doll et al. (2019), Li et al. (2022)	The generation of novel, functional gene sequences (including cis- or transgenes) via the generation of multiple contiguous mutations ×
Base editing is emerging as a more facile method to generate targeted SNPs in a specific diploid or polyploid parental genotype. Unlike SDN1/2/3, these do not depend on Non-Homologous End Joining or Homologous Recombination and may be homozygous in the first generation.	Targeted nucleotide substitution	Bharat et al. (2020), Molla et al. (2021)	Base edit at a single pre-determined genomic location ✓✓✓	Substitutions of DNA bases occur spontaneously throughout the genome. As above, screening for individuals possessing equivalent mutations is facile in some crops. Traditional methods to combine multiple mutations at different loci using marker assisted selection is also possible in some crop species but more challenging or impossible in vegetatively propagated, perennial, self-incompatible etc crops. The generation of de novo gene sequences by spontaneous substitution and iterative selection of multiple contiguous bases is effectively impossible using current traditional breeding approaches. However, the introgression of one or multiple existing cisgenes is relatively facile.	Wang et al. (2020)
		Li et al. (2017)	Base edits at several, independent pre-determined genomic locations in same plant ✓
			Multiple base edits in contiguous nucleotide positions to generate a completely novel gene ×
SDN-3 / cisgenics Single or multiple site-directed nucleases with repair template can produce targeted and homozygous whole gene level changes in an elite diploid or polyploid parental genotype to obviate the need for repeated backcrossing.	Allele (cisgene) replacement or de novo cisgene addition	Li S. et al. (2016); Schmidt S.M. et al. (2020); Jo et al. (2014)	Single allele (cisgene) replacement or de novo cisgene addition ✓✓✓	Sexual crossing results in novel combinations of alleles. Screening for individuals possessing a specific allele is relatively facile using molecular markers. Traditional methods to combine specific alleles at multiple loci using marker assisted selection is also possible in some crop species but more challenging or impossible in vegetatively propagated, perennial, self-incompatible etc crops. Repeated backcrossing to a recurrent parent combined with MAS can result in a single or multiple allele replacement.	Chung et al. (2017); Cho et al. (2019)
			Multiple allele (cisgene) replacements or de novo cisgene transfers into the same plant ✓
	Targeted insertion of multiple cisgenes or transgenes at a single locus using gene editing	Gao et al. (2020)	Simultaneous insertion of multiple genes into a single, segregating locus ×?	The introgression of one or more cisgenic alleles or the repeated, iterative stacking of cisgenes into untargeted locations is possible. However, introgressing multiple cisgenes into a single, predefined, genomic landing locus is effectively impossible using current traditional breeding approaches, However, the function of the individual cisgenes is unlikely to be significantly altered by their genomic location.
	Trait stacking into a predetermined genomic locus	Ainley et al. (2013)	Iterative stacking by adding new genes to an already present and pre-determined ‘safe harbour’ / ‘landing pad’ locus ×?