Table 1.

Recent publications using DNA-free Genome Editing approaches.

Reference	Plant species	Trait	GE-technique	Tissue	Delivery	Editing efficiency	Off-targets	Method
Woo et al., 2015	Arabidopsis thaliana, Lactuca sativa, Nicotiana attenuata, Oryza sativa	POC	CRISPR/Cas9 RNPs	Protoplasts	PEG-fusion	Calli: Monoallelic mutations: 5.7%; Biallelic mutations: 40%	0/104 (2–5 MM)	Targeted deep seq.
Baek et al., 2016	Chlamydomonas reinhardtii	Yield, abiotic stress	CRISPR/Cas9 RNPs	Single cells	Electroporation	Cells: Up to 0.56%	0/17 (2–4 MM)	Targeted deep seq.
Malnoy et al., 2016	Malus domestica, Vitis vinifera	Biotic stress	CRISPR/Cas9 RNPs	Protoplasts	PEG-fusion	Protoplasts: 0.1–6.9%	n.d.	n.d.
Stoddard et al., 2016	Nicotiana benthamiana	POC, herbicide resistance	TALEN mRNA	Protoplasts	PEG-fusion	Protoplasts: No-UTR DNA control: 70.5%, No-UTR mRNA control: 5.8%	n.d.	n.d.
Shin et al., 2016	C. reinhardtii	POC	CRISPR/Cas9 RNPs	Single cells	Electroporation	Up to 3 × 10-5	0/333 (1–3 MM)	WGS
Svitashev et al., 2016	Zea mays	Male sterility, herbicide tolerance	CRISPR/Cas9 RNPs	Immature embryos	Biolistics	Embryo: Cas9 only: 0.002–0.02 DNA delivery: 0.18–0.56 RNP delivery: 0.01–0.69	n.d.	n.d.
Subburaj et al., 2016	Petunia hybrid	Herbicide resistance	CRISPR/Cas9 RNPs	Protoplasts	PEG-fusion;	Protoplasts: Cas9 protein: 0.03–27.13, NR-RGEN: 5.3–34.69	n.d.	n.d.
Zhang et al., 2016	Triticum aestivum	Yield	CRISPR/Cas9 mRNA	immature embryos	Biolistics	No. of transgen-free frequency: 0–100% No. of homozygous transgen-free frequency: 0–36.8%	0/8 (2–4 MM)	PCR- RE
Ferenczi et al., 2017	C. reinhardtii	POC, Gene replacement	CRISPR/Cpf1 RNPs	Single cells	Electroporation	Colonies: 0.12–16%	n.d.	n.d.
Grahl et al., 2017	Non-albicans candida	POC	CRISPR/Cas9 RNPs	Single cells	Electroporation	n.d.	n.d.	n.d.
Kim et al., 2017	Glycine max, N. attenuata	Fat-synthesis; POC	CRISPR/Cpf1 RNPs	Protoplasts	PEG-fusion	Soybean: Protoplasts: LbCpf1: 0–11.7% AsCpf1: 0–1.6% Tobacco: LbCpf1 and AsCpf1: < 0.1% LbCpf1 + crRNA and AsCpf1 + crRNA: < 1%	0/23 (4–6 MM)	Targeted deep seq.
Liang et al., 2017	T. aestivum	Yield	CRISPR/Cas9 RNPs	Protoplasts; immature embryos	PEG fusion, Biolistics	Protoplasts: 33.4 and 21.8% Embryo cells: gw2-RNPs: 0.18–0.21% pGE-TaGW2: 0.99–1% gasr7-RNP: 45.3%	0/20 (2–5 MM)	Sanger seq.
Andersson et al., 2018	Solanum tuberosum	Starch syntheses	CRISPR/Cas9 RNPs	Protoplasts	PEG-fusion	Protoplasts: 1–25%	n.d.	n.d.
Liang et al., 2018	T. aestivum, O. sativa	Yield/ POC	CRISPR/Cas9/Cpf1 RNPs, TALEN-Proteins	Protoplasts; immature embryos	PEG fusion, Biolistics	Protoplasts: 10.9–33.6%	n.d.	n.d.

GE-Technique, Genome editing technique; RNPs, Ribonucleoproteins; WGS, Whole Genome sequencing; POC, Proof of concept; n.d., not determined.