Table 3.

Mutagens applied for induction of target mutations in rice.

Method	Mechanism of mutation	Mutation frequency	Mutants produced	Pros	Cons
MN	Generates short 3’ overhangs, predominantly result in larger deletions, with occasional larger insertions.	Has not been applied to rice		MNs generate deletions, which range in size from 2 to 71bp in maize.	Native MNs targeted sequences are limited; Introduction of specificities are difficult because the DNA-binding and endonuclease activities are on the same domain.
ZFN	Predominantly small deletions, but a larger proportion of insertions than TALENs.	−	Identification of safe harbor loci (Cantos et al., 2014);	ZFNs are easy to design, due the preexisting Zinc Fingers known recognition patterns.	ZFNs produce more insertions than TALENs, it was proposed as a disadvantage if engineered cereals are taken through the regulatory approval process.
			Plant development and metabolism (Jung et al., 2018);
			Industrial quality (Jung et al., 2018);
TALEN	Predominantly small deletions and occasionally insertions and substitution.	calli T0 (4-30%) (reviewed in Zhu et al., 2017)	Biotic stress resistance (Li et al., 2012);	TALENs bind with greater specificity than ZFNs.	Construction of the DNA recognition motif is laborious; Require a thymine at the first position.
			Industrial quality (Ma et al., 2015; Shan et al., 2015)
			Herbicide resistance (Li et al., 2016b)
CRISPR/Cas9	Small indels (≤10pb), often single nucleotides, inserts mostly A/T base pairs and deletions.	85%-100% (reviewed in Zhu et al., 2017)	Yield and quality (Li et al., 2016d; Li et al., 2016e; Xu et al., 2016; Zhou et al., 2016; Miao et al., 2018; Sun et al., 2018; Barman et al., 2019; Li et al., 2019; Pérez et al., 2019)	High specificity through gRNA.	Undesirable off-targets, PAM requirement for AT residues, high quantity of mismatches tolerance.
			Plant development and metabolism (Li et al., 2017c; Cheng et al., 2019; Lee et al., 2019b)
			Nutritional quality (Sun et al., 2017; Songmei et al., 2019; Yang et al., 2019)
			Biotic stress resistance (Zhou et al., 2015; Wang et al., 2016; Macovei et al., 2018; Li et al., 2019)
			Abiotic stress tolerance (Xu et al., 2014; Sun et al., 2016; Zhang et al., 2019)
CRISPR/Cpf1 (Cas12)	Generate 5’ overhangs which predominantly result in deletions.	47.2% (Endo et al., 2016)	Plant development and metabolism (Hu et al., 2017; Tang et al., 2017; Wang et al., 2017; Yin et al., 2017)	High specificity through crRNA and less frequency of off-targets.	PAM requirement for GC residues.