Table 1.

gRNA modifications to improve CRISPR-Cas9 efficiency in cultured mammalian cells.

Modification(s)	Modification location	Effect on genome editing efficiency	References
M	Terminal residues	↑#	Hendel et al., 2015a; Rahdar et al., 2015
MS	Terminal residues	↑#	Hendel et al., 2015a; Basila et al., 2017; Finn et al., 2018
	Spacer (PAM-distal region)	↑*	Yin et al., 2017; Finn et al., 2018; Mir et al., 2018
	Spacer (tracrRNA-binding region)	↑*	Yin et al., 2017; Finn et al., 2018; Mir et al., 2018
	Spacer (Seed region)	↓	Yin et al., 2017; Mir et al., 2018
MSP	Terminal residues	↑#	Hendel et al., 2015a
cEt	Spacer (PAM-distal region)	↑	Rahdar et al., 2015
	Spacer (tracrRNA-binding region)	↑	Rahdar et al., 2015
	Spacer (Seed region)	↓	Rahdar et al., 2015
2′-F	Spacer (PAM-distal region)	↑	Rahdar et al., 2015
	Spacer (tracrRNA-gbinding region)	↑	Rahdar et al., 2015
	Spacer (Seed region)	↓	Rahdar et al., 2015; O'Reilly et al., 2019
2′-F + PS	Spacer (PAM-distal region)	↑	Yin et al., 2017; Mir et al., 2018
	Spacer (tracrRNA-binding region)	↑	Yin et al., 2017; Mir et al., 2018
	Spacer (Seed region)	↓	Yin et al., 2017; Mir et al., 2018
	Spacer (Seed region, Cas9-non-interacting residues)	↑*	Yin et al., 2017; Mir et al., 2018
PS	Whole crRNA	↑	Rahdar et al., 2015

^*additionally validated in vivo.

^#additionally validated in human primary cells.

2′-O-methyl (M or 2′-O-Me); 2′-O-methyl 3′phosphorothioate (MS); 2′-O-methyl-3′-thioPACE (MSP); S-constrained ethyl (cEt); 2′-fluoro (2′-F); and phosphorothioate (PS).