Massively parallel single-amino-acid mutagenesis

Abstract

Random mutagenesis methods only partially cover the mutational space and are constrained by DNA synthesis length limitations. Here we demonstrate programmed allelic series (PALS), a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing.

Figure 1

Programmed Allelic Series (PALS) mutagenesis in a single volume reaction. (a) Primers are synthesized in parallel on a microarray, tiling a target sequence of interest and bearing programmed mutations (“X”), e.g., to make specific or random codon substitutions or tiling deletions. Programmed mutations are introduced by primer extension on a degradable wild-type template (marked with deoxyuracil, “U”) followed by PCR amplification with primers directed to the gene flanks (black) or to adaptor sequences within the mutagenized strands (brown). A final PCR step yields full-length copies incorporating a single programmed mutation per copy. (b) Mutant libraries are cloned, with each clone receiving a unique molecular tag sequence. The library is subjected to hierarchical shotgun sequencing, with paired end reads interrogating the target gene insert from one end and the molecular tag from the other, to yield a set of consensus haplotypes and associated tags.

Figure 2

En masse functional selection of Gal4 DBD PALS library highlights residues and mutations critical for transcriptional activity. Sequence-function maps of mutation effect sizes across Gal4 DBD residues 2-65 (rows) for all programmed amino acid substitutions (columns; STOP: premature stop codon, Δ: in-frame codon deletion) following outgrowth either without selection (upper: SC – uracil, after 24 h) or under stringent selection for Gal4 (lower: SC – uracil – histidine + 1.5 mM 3-AT, after 64 h). Sequence-function maps are shaded by the log2-effect size for each residue and substitution, ranging from improved growth versus wild-type (red), equivalent to wild-type (white), to slower growth than wild-type (blue). Yellow and gray boxes denote the wild-type residue or insufficient data, respectively (minimum four distinct tagged haplotypes per codon substitution required in the non-selective library). Below, evolutionary conservation among Zn2/Cys6 family members (plotted in bits), confirms selective constraint to maintain the six domain-defining cysteines (indicated by arrows).