Table 1 Principle and mutational scope of a selected range of methods focused on the generation of libraries that tolerate length and compositional variation.

COBARDE¹¹

Orthogonal protecting groups in oligonucleotide synthesis. One protecting group is used in the high efficiency synthesis of the oligonucleotide while a second one, introduced stochastically, allows synthesis to be interrupted. Libraries contain only deletions from a maximum length synthesis program. Deletions can vary in length and depend only on the frequency and concentration of stochastically used protecting group

Codon deletion mutagenesis (CDM)¹³

Engineered asymmetric Mini-Mu transposon random integration followed by Type IIS restriction endonuclease cleavage and target re-ligation. Transposon sequence (position of Type IIS recognition site) determines size of deletion, ranging from three to 15 nucleotides. In frame deletions can be selected ensuring only deletions are introduced in library

MuDel¹⁶

Mini-Mu transposon random integration followed by Type IIS restriction endonuclease cleavage and target re-ligation. Transposon excision removes three nucleotides, creating a codon-length deletion but not necessarily in frame. Mutations therefore include deletions as well as standard mutations in neighbouring residues

Pentapeptide scanning mutagenesis (PSM)¹⁵

Tn4430 transposon random insertion followed by orthodox Type II restriction endonuclease cleavage and target re-ligation. The use of orthodox endonucleases creates fixed “scar” sequences on the DNA level that limit the diversity of the inserted pentapeptide. Method is limited to 5-residue insertions

RAISE¹⁸

Terminal transferases (TdT) are used to introduce random sequences at the end of dsDNA fragments of the target DNA. Self-priming PCR is subsequently used to reassemble the target DNA incorporating TdT-generated material. Libraries introduce both sequence and length variation randomly in the library which can include both deletions and insertions

Random Elongation mutagenesis (REM)¹⁹

Short double-stranded DNA fragments containing sequence diversity are introduced via orthodox Type II restriction endonucleases to create terminal in frame insertions. The use of orthodox Type II restriction endonucleases introduces fixed “scar” sequences in generating a library. Selection of the dsDNA fragment offers control over the diversity and length of the inserted sequence

RID mutagenesis¹⁷

Circularized single-stranded target DNA is chemically cleaved to linearise them. Adaptor oligonucleotides (harbouring Type IIS restriction endonuclease sites) are ligated to the cleaved target DNA and PCR amplified. Circularization and removal of adaptor sequences result in a concomitant 3-base deletion (from the target) and an insertion (controlled by the sequence in the adaptors)

TRIAD¹⁴

The method expands on the MuDel approach introducing adaptors after the restriction endonuclease removal of transposon sequences which lead to larger deletions (up to 3 residues) or insertions (up to 3 residues). Mutations need not be in frame and adaptors define the sequence being inserted (and can include sequence variation)

TRINS²⁰

Circularized single-stranded DNA fragments of the intended target are used as templates for limited rolling circle amplification. PCR assembly of fragments generates libraries of the target genes containing random tandem repeats of gene sequences. TRINS libraries reported show a large variation in insertion lengths, which need not be in frame

InDel Assembly (this study)

Additive synthesis of the target DNA using building blocks that are stochastically incorporated in the reaction. Mutations include deletions from a maximal length assembly but can introduce sequence variation by mixture of building blocks. While building blocks presented here are three nucleotide long, the length of the building blocks can be varied, enhancing the library complexity that can be obtained