Table 2.

A summary of the mutagenesis methodologies outlined in this review. Efficacy is defined as the proportion of transformant cells encoding any mutations. Efficiency is defined as the proportion of the mutated transformant cells with all desired sequences or mutations. NR highlights where data is not reported in the original publication.

Category	Method	Features	Efficacy	Efficiency	Reference
Restriction enzyme-based	Type IIS restriction enzyme (BsaI) modular assembly	Mix-and-match assembly of variant gene fragments via BsaI digestion and DNA ligation, similar to Golden Gate cloning. Limited to small library sizes.	100%	n.a.	(Popova et al., 2015; Quaglia et al., 2017)
	VersaTile	Mix-and-match assembly of protein modules, based on Golden Gate assembly.	95%	n.a.	(Gerstmans et al., 2020)
Ligation-based	Darwin assembly	Mutations encoded by oligonucleotides are incorporated by DNA polymerase and ligase activities. Method has high efficiency for multiple mutation sites.	98–100%	100%	(Cozens and Pinheiro, 2018)
	ProxiMax	Ligation of defined codons to build a library without codon bias. Requires trimer oligonucleotides, robust and controllable but becomes challenging for multiple mutation sites.	NR	100%	(Ashraf et al., 2013)
PCR-based	QuikChange (and variations thereof)	Mutations introduced by a primer pair during inverse PCR. Limited to mutation of one position.	84%	55%	(Mao et al., 2011; Xia et al., 2015)
	Overlap extension PCR (OE-PCR)	A gene fragment is amplified by PCR (with primers adding both mutations and homologous termini), then the full-length sequence is assembled by overlap extension PCR. Method is robust and reliable, though cumbersome for multiple mutation sites.	>90%	100%	(An et al., 2005; Bryksin and Matsumura, 2010; Cheng et al., 2017; Heckman and Pease, 2007; Hussain and Chong, 2016; Wäneskog and Bjerling, 2014; Wei et al., 2012; Williams et al., 2014; Xiao and Pei, 2011)
	Asymmetric PCR	A single-stranded gene fragment is amplified by asymmetric PCR using mutagenic primers, which is then used as a megaprimer to introduce mutations into full sequence. Method is robust and reliable, though cumbersome for multiple mutation sites.	91–100%	100%	(Bi et al., 2012; Sadler et al., 2018)
	SpeedyGenes	Gene synthesis method, encoding mutations using the overlapping oligonucleotide primers that assemble the gene library de novo. Many combinatorial mutations can be efficiently assembled, though efficiency drops for large genes.	76–90%	100%	(Currin et al., 2017, Currin et al., 2014)