Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

Table 1 Summary of CRISPR/Cas genome editing assays in plants

Material/activity	*A. thaliana*	*N. benthamiana*	*O. sativa*	*T. aestivum*	*S. bicolor*	Reference
Cas9
Codon-optimized for plants	Yes	Yes	Yes	Yes	Yes	[10, 11, 15, 16]
Codon-optimized for plants	No	No	No			[9, 12–14, 16]
Number of NLS	2	2	2	2		[10–14]
		1				[9, 16]
	1		1		N/A	[16]
			1			[7, 8, 15]
Intron introduced	Yes	Yes				[11]
Intron introduced	No	No	No	No	No	[9, 10, 12–16]
Promoter	35S PPDK	35S PPDK				[11]
	2x CaMV 35S		2x CaMV 35S	2x CaMV 35S		[10, 12]
	CaMV 35S	CaMV 35S	CaMV 35S			[9, 11, 14, 16]
					OsAct1	[16]
	AtUBQ		OsUBQ			[13]
			ZmUBQ			[15]
sgRNA
Promoter	AtU6	AtU6				[9, 11–13, 16]
			OsU3			[10, 12–15]
			OsU6		OsU6	[16]
				TaU6		[10]
Assays
	AtPDS3		OsPDS
	AtFLS2		OsBADH2
	AtBRI1		OsMPK2
	AtJAZ1		Os02g23823
	AtGAI		OsROC5
	AtCHL1		OsSPP
Genes targeted	AtCHL2	NbPDS	OsYSA	TaMLO	N/A	[9–16]
	At5g13930		OsMYB1
			OsMPK5
			OsCAO1
			OsLAZY1
			OsSWEET11
			OsSWEET14
Transient assays
Protoplasts	Yes	Yes	Yes	Yes	No	[10–14, 16]
NHEJ mutation frequency	1.1-5.6% [11]	37.7-38.5% [11]	14.5-38% [10]	28-29% [10]
			3-8% [14]
HDR modification frequency	18.8% [12]*	9% [11]	6.9% [10]
	42% [13]*
Leaf agroinfiltration	Yes	Yes	No	No	No	[9, 11, 16]
NHEJ mutation frequency	2.7% [11]	4.8% [11]
		2.1% [9]
Embryo transformation	No	No	No	No	Yes	[16]
NHEJ mutation frequency					28%
Transgenic mutants
Mutated plants recovered	Yes	Yes	Yes	N/A	N/A	[9, 10, 12, 13, 15]
Frequency of modified plants	30-84% [12]	6.7% [9]	4-9.4% [10]
	76-89% [13]		5-75% [12]
			50% [13]
			83-91.6% [15]
Biallelic mutations recovered	Yes [12, 13]	No [9, 11]	Yes [10, 12, 14, 15]	N/A	N/A
Off-targets
Off-target detected	N/A	No [9]	Yes [10, 14]	N/A	N/A	[9, 10, 14]

*In YF-FP assays.
Abbreviations: A. thaliana: Arabidopsis thaliana, N. benthamiana: Nicotiana benthamiana, O. sativa: Oryza sativa, T. aestivum: Triticum aestivum, S. bicolor: Sorghum bicolor.

ISSN: 1746-4811