Table 2.

Methodological details for diverse saturation mutagenesis studies of protein function

Cell Type	Protein/Gene name	Library size	Mutagenesis and screening methodologies	Ref
Bacterial	TEM-1 β-lactamase	287 positions, 3 libraries in single mutant backgrounds with 5434 mutants in each	Pfunkel mutagenesis, selection on various antibiotic concentrations	[19]
Bacterial	APH kinase	264 positions, 4234 mutants	MITE (Mutagenesis by Integrated TilEs), growth selection on various aminoglycosides	[16]
Bacterial	Ras	165 residues, 2 libraries in WT and single mutant backgrounds	Mutagenesis using partially overlapping primers, screening by bacterial two-hybrid system	[31]
Yeast	Gal4	64 positions, 1196 mutants	PALS (Programmed allelic series) mutagenesis, screening based on yeast two-hybrid system	[13]
Yeast	Hsp82 ATPase domain	219 positions, 4021 mutants	EMPIRIC methodology, growth rate screening	[9]
Mammalian	BCR-ABL1 kinase domain	20 positions, 380 mutants	CRISPR-Cas9-based genome editing approach, fluorescence- based screening by bulk competition of murine BalF3 cells	[55]
Bacterial	GFP	51715 protein variants Upto 15 mutations	Random mutagenesis, FACS based screening	[34]
Yeast	BH3 peptides	1026 variants	Synthetic peptides, screened by SORTCERY (FACS screening and gating strategy that rank orders variants based on their relative counts in bins sorted based on affinities)	[56,57]
Mammalian	Ubiquitin fused to EGFP	N- terminal residues	Landing pad cell line was developed to transfer libraries of mammalian genes into the mammalian genome	[58]