Table 2.

Performance overview of double‐ and multi‐site saturation mutagenesis methods (e. g. for targeted protein engineering).

Method	Library size	% Coverage	% 1 NSM	% 0 NSM (wild type)	% 2 NSM	% >2 NSM	Ref.
Double‐site mutagenesis
Nicking mutagenesis (NM)	n/a	79.2	n/a	60.0	n/a	n/a	[36]
Plasmid recombineering	5940	98.0	26.3	32.6	24.5	16.4	[41]
Multi‐site mutagenesis
Optimized multi‐site nicking mutagenesis (NM)[a]	16 384	99.9	n/a	2.58	n/a	n/a	[49]
	32 768	99.4	n/a	0.25	n/a	n/a	[49]

n/a: not available; [a] The average of the two reported independent runs of optimized multi‐site NM is given (Table 1 in Ref. [49]).