Table 2.

Schematic summary of the strategies employed to generate HE variants with novel target specificities

Method	Cluster mutagenesis	Directed evolution	Chimeras	Single-chain variants	Computational redesign	Semi-rational design	Semi-rational genomic target design
Homing Endonuclease	I-CreI I-CreI I-CreI I-Ssp6803I	I-SceI PI-SceI I-ScaI PI-SceI I-SceI	I-DmoI_I-CreI I-DmoI_I-CreI PI-SceI I-DmoI	I-CreI I-MsoI I-CreI	I-MsoI I-CreI	I-CreI	I-CreI
Structural information required	Yes Yes Yes Yes	No Yes Yes	Yes Yes Yes Yes	Yes Yes Yes	Yes Yes	Yes Yes	Yes Yes Yes
Variant selection	Two-plasmid selection in E. coli High-throughput fluorescence competition-binding assay	Two-plasmid (two-hybrid) selection in E. coli	In vitro cleavage	In vivo cleavage Two-plasmid selection in E. coli In vitro and in vivo cleavage and reparation	In vitro cleavage	High-throughput screening in yeast	Combinatorial generation of variants and high-throughput screening of variant/target in yeast
Variant/DNA crystal structures	No Yes Yes Yes	No No No	Yes No No No	No Yes Yes (Unpublished)	Yes No	No No	No No Yes
References	[164] [165] [166] [99]	[167] [168] [125]	[25] [26] [169] [170]	[26] [171] [27]	[128] [172]	[126] [36]	[173] [35] [38]