. 2020 Nov 21;9(11):2518. doi: 10.3390/cells9112518

Table 3.

Comparison between different fdCas9 constructs available in the literature.

	sgRNA Delivery Method	Linker	Gene Editing Activity of fdCas9			Optimal Spacer Distance	Off-Targets
	sgRNA Delivery Method	Linker	Compared to Negative Control	Compared to Single WT Cas9	Compared to Paired Casas9 nickases	Optimal Spacer Distance	Genes Tested	Activity
Tsai (2014) [22] (Figure 2A)	Csy4-based dual sgRNA expression system	GGGGS linker	3–40%	Differences varied depending on gene tested	Similar to fdCas9	13–18 bp	VEGFA	Indistinguishable off-target mutation
Guilinger (2014) [23] (Figure 2B)	dual sgRNA expression plasmid	17 linkers tested; best activity using XTEN linker	GFP disruption: 10% reported by flow cytometry and 20% by T7EI	eGFP disruption: 25% reported by flow cytometry and 2/3 of fdCas9 activity by T7EI	eGFP disruption: 15% reported by flow cytometry and similar activity to fdCas9 by T7EI	15 and 25 bp	AAVS1, CLTA, EMX1, HBB, VEGFA	Fold increase of on/off-target editing: 140 compared to WT Cas9 1.3–8.8 compared to nickases
Guilinger (2014) [23] (Figure 2B)	dual sgRNA expression plasmid	17 linkers tested; best activity using XTEN linker	Average of 14.9% on human genes	Average of 28.2% on human genes	Average of 20.6% on human genes	15 and 25 bp	AAVS1, CLTA, EMX1, HBB, VEGFA
Nakagawa (2015) [25] (Figure 2C)	All-in-one construct, included in the fdCas9 plasmid	GGGGS linker	Tested on mice fertilized oocyte			13–18 bp	Top three candidates of used sgRNA	No off-target editing was reported
Nakagawa (2015) [25] (Figure 2C)	All-in-one construct, included in the fdCas9 plasmid	GGGGS linker	Average of 49%, and moderate birth rate	Average of 90%, and a low birth rate	Average of 2.9%, and a high birth rate	13–18 bp	Top three candidates of used sgRNA	No off-target editing was reported
Aouida (2015) [24] (Figure 2D)	sgRNA expressing DNA fragments	XTEN linker	eGFP disruption: 5% reported by flow cytometry	eGFP disruption: 12.3% reported by flow cytometry	eGFP disruption: 1% reported by flow cytometry	15–39 bp	CCR5, AAVS1, EMX1, HBB	Only WT Cas9 showed 25–30% off-target editing
Havlicek (2017) [26] (Figure 2E)	Csy4-based multiple sgRNA expression systems	(GGGGS)₅	Average of about 30% human gene editing	Cas9 orthologs including SpCas9-HF1 and eSpCas9 showed higher activity	Not tested	13–18 and 26 bp	CLTA, EMX1, VEGFA	fdCas9 outperform limited off-target editing compared to all Cas9 orthologs tested

GFP- green fluorescence protein, AAVS1- Adeno-Associated Virus Integration Site 1, CLTA- Clathrin Light Chain A, EMX- Homeobox protein 1, HBB- Hemoglobin Subunit Beta, VEGFA- Vascular Endothelial Growth Factor A, CCR5- chemokine receptor 5.