Table 3.

Optimization of CRISPR/Cas9 system

	Cas9		gRNA
Improvement	Refs.	Improvement	Refs.
Efficiency	Codon-optimized Cas9	[29], [31], [50]	Modification on 3′ end of crRNA:tracrRNA chimera	[31]
St1Cas9 and SaCas9	[28], [51]	Csy4-based gRNA cleavage	[54], [55]
Cpf1	[52]	Chemically-modified gRNAs	[56]
Specificity	Cas9 nickase	[57], [58]
eSpCas9	[59]
FokI-based CRISPR/Cas	[55], [60]
Cas9 activity	dCas9-KRAB and dCas9-VP160	[69]
Photoactivatable dCas9	[66]
Spatiotemporal control	Specific promoter driven Cas9	[47]
Doxycycline-regulated Cas9	[62], [63], [64]
Rapamycin-inducible split-Cas9	[65]
Photoactivatable Cas9	[66]

Note: Cas9, CRISPR-associated protein 9; St1Cas9, Streptococcus thermophilus Cas9; SaCas9, Staphylococcus aureus Cas9; Cpf1, Cas protein 1 of PreFran subtype; eSpCas9, “enhanced specificity” Streptococcus pyogenes type II Cas9; dCas9, catalytically inactive Cas9; KRAB, Krüppel associated box; gRNA, guide RNA; crRNA, CRISPR RNA; tracrRNA, trans-activating crRNA; Csy4, CRISPR subtype Ypest protein 4.