Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

Abstract

Next-generation sequencing has engendered an expanding suite of functional assays that can test sequence-function relationships at unprecedented scales in pooled formats (multiplex). Such assays are currently constrained by the short length of oligonucleotide (oligo) pools, which limit potential applications. Here we report a simple, low-cost, and scalable method called DropSynth that assembles gene libraries from oligo pools for use in multiplexed functional assays. DropSynth utilizes a library of barcoded beads to isolate and concentrate oligos needed for a gene's synthesis in a pooled format. These bead-bound oligos are then emulsified, processed, and assembled into genes within the emulsion droplets. We synthesized~1000 phylogenetically diverse orthologs of phosphopantetheine adenylyltransferase (PPAT) and tested their fitness in a multiplexed functional assay. While the majority of orthologs complement, those that do not are broadly distributed across the phylogenetic tree. Synthetic errors in our assemblies allow us to explore local landscapes around the designed orthologs revealing constrained mutations for complementing orthologs as well as gain-of-function mutations for low-fitness orthologs. This broad mutational scanning approach is complementary to deep mutational scanning and helps us understand proteins by probing evolutionarily divergent sequences that share function.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/163550 doi: bioRxiv preprint first posted online Jul. 14, 2017;The rapid progress in DNA sequencing has provided a glimpse of the vast diversity of sequence and function that nature has explored. Unfortunately our ability to explore the functional aspects of diverse sequences remains minuscule. ) that link a function to a next generation sequencing (NGS)-based output to allow testing of thousands to millions of sequences for function in a pooled manner. Mutagenesis and deep mutational scanning combined with multiplexed functional assays are now able to systematically probe all single and double amino acid substitutions for a protein's function 3,4 . Despite the power of these approaches, the sequence space explored in such experiments is minute when compared to the evolutionary distance between even highly-homologous protein sequences. Our ability to sample a broad diversity of sequences is bottlenecked by two limitations in existing approaches for producing the libraries to be tested. First, low-cost microarray-based oligo libraries allow for large libraries of designed <200 nucleotide (nt) sequences 5 , which is far below the typical length of a protein and limits many other applications. Secondly, gene synthesis is capable of creating long-length sequences, but its costs currently make it prohibitively expensive for sampling large libraries of designed sequences [6][7][8][9] .Here we develop a new gene synthesis method we term DropSynth, a multiplexed...

CasPER, a method for directed evolution in genomic contexts using mutagenesis and CRISPR/Cas9

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“…15). As oligonucleotide and gene library synthesis improves, we expect to include additional genetic context in the assays 49,50 .…”

Minimum epistasis interpolation for sequence-function relationships

“…ecent advances in quantification via next-generation sequencing have allowed the proliferation of high-throughput combinatorial mutagenesis assays that measure molecular function for tens of thousands to millions of sequences simultaneously 1 . These assays have been applied to many different classes of functional elements, including protein-coding sequences [2][3][4][5][6][7][8][9][10][11][12][13] , RNAs [14][15][16][17][18] , and regulatory or splicing elements [19][20][21][22] . However, in practice, due to both the vastness of sequence space and the limitations of techniques for library preparation, such experiments typically result in missing measurements for a subset of possible genotypes.…”