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A library containing approximately 5000 afxynG1 mutants was generated and thermally screened at 60° C for 30 min.

To identify novel mutation avoidance genes in mycobacteria, a M. smegmatis mc2 155 library of ∼11,000 independent transposon insertion mutants was generated and screened for spontaneous mutations that confer rifampicin resistance (Rif-R), used as a hypermutator hallmark (Fig. 1a).

A CP library of 97 mutants was generated to identify the effect of CP on three characteristic regions of RhEst1 including the flexible cap domain (Region 1), the region around the entrance to substrate binding pocket (Region 2) and the surface-exposed sectors in catalytic domain (Region 3).

Therefore, two different PCR conditions to yield medium mutation frequency (MMF: 4 9 mut/kb) and high mutation frequency (HMF: 9 16 mut/kb) were adopted in this work, and a library of 5000 MMF and 5000 HMF mutants was generated.

A Pbs2 random mutagenesis library (9×104 mutants) was generated with a Stratagene kit and cloned into the pRS416-GPD1 vector.

To systematically address the role of the partially exposed N-terminus in BSX stability, a series of mutants was generated in which the first hydrophobic residue, valine (Val1), was either deleted or substituted with various amino acids.

Mutants were generated using the Takara MutanBEST Kit.

K3036.58E MOP receptor and E2976.58K KOP receptor mutants were generated.

The ttr-11 km64 ttr-11 km64(km85) deletion mutands ttr-57 km85 ttr-57 km85e CRISPR-Cas9 system41.

HLA-A2 mutants were generated using overlap extension polymerase chain reaction.

shRNA-resistant TFEB mutants were generated using site-directed mutagenesis (Q5 0552S, New England Biolab).