Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes

Abstract

SUMMARY We describe a multiplex genome engineering technology in Saccharomyces cerevisiae based on annealing of synthetic oligonucleotides at the lagging strand of DNA replication. The mechanism is independent of Rad51-directed homologous recombination and avoids the creation of double-strand DNA breaks, enabling precise chromosome modifications at single base-pair resolution with efficiencies >40% without unintended mutagenic changes at the targeted genetic loci. We observed the simultaneous incorporation of up to 12 oligonucleotides with as many as 60 targeted mutations in one transformation. Iterative transformations of a complex pool of oligonucleotides rapidly produced large combinatorial genomic diversity >105. This method was used to diversify a heterologous β-carotene biosynthetic pathway that produced genetic variants with precise mutations in promoters, genes, and terminators, leading to altered carotenoid levels. Our approach of engineering the conserved processes of DNA replication, repair, and recombination could be automated and establishes a general strategy for multiplex combinatorial genome engineering in eukaryotes.

Biosystems design by directed evolution

“…In these two methods, homologous recombination was also introduced through the λ‐Red recombineering system but used double‐strand DNA as substrates. MAGE was also adapted for use in S. cerevisiae , such as yeast oligo‐mediated genome engineering (YOGE) and eukaryotic MAGE (eMAGE) . MAGE and its related methods have been successfully applied in directed evolution on the genome‐scale .…”

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

“…Finally, at present, most of the CRISPR/Cas9-based directed evolution methods rely on integration of relative small (80-120 bp) DNA fragments (Barbieri et al, 2017;Garst et al, 2017;Guo et al, 2018) and may require time-consuming construction of donor variant libraries and/or relative costly DNA synthesis of diversified array-based oligos (Nyerges et al, 2018;Roy et al, 2018). With CasPER, multiplex genome engineering of larger genomic loci is now demonstrated at very high efficiencies along the full size of donor fragment lengths, and should thereby enable cost-effective, high-throughput and robust evolution studies of even complex multi-genic traits in any organism supporting genome integration of heterologous DNA by homologous recombination.…”

“…Cas9 is an RNA-guided endonuclease that introduces double-stranded breaks (DSBs) at almost any target DNA locus where a protospacer-adjacent motif (PAM; NGG) is present, yielding several hundred thousand genomic target sites in an average eukaryotic genome (DiCarlo et al, 2013). With the ability to target and integrate heterologous DNA fragments at high efficiencies by way of homologous recombination, several studies have investigated directed evolution and sequence-function relationships of regulatory and genic regions in genomic contexts, albeit using short oligonucleotides as repair donors (Barbieri et al, 2017;Findlay et al, 2014;Garst et al, 2017;Storici et al, 2001;Wang et al, 2009). In addition, larger mutagenized DNA fragments were in vivo assembled and integrated into the S. cerevisiae genome by employing Cas9 and a selection marker (Ryan et al, 2014).…”

Recent insights into the genotype–phenotype relationship from massively parallel genetic assays

“…Scaling up deep mutational scanning experiments to the scale of the genome is at present out of reach: bottlenecks include the precise, genome-wide introduction of individual mutations (mutagenesis efficiency and accuracy), sequencing costs and associating mutations at distant loci. The first is improving with advances in genome engineering, particularly from CRISPR-Cas9-based methods (Barbieri, Muir, Akhuetie-Oni, Yellman, & Isaacs, 2017;Haimovich, Muir, & Isaacs, 2015;Roy et al, 2018). The second is continuing to improve, following a long-term trend of decreasing costs (but see (Muir et al, 2016) for the alternative challenge of managing increasing amounts of data); and the third is becoming more feasible with emulsion-based generalized DNA assembly technologies that encapsulate single cells and enable distal DNA sites to be linked by sequencing (either by directly ligating mutated sites adjacent to each other (Haliburton, Shao, Deutschbauer, Arkin, & Abate, 2017;Zeitoun et al, 2015) or, more scalably, by ligating them to a cell-specific DNA barcode (Zeitoun, Pines, Grau, & Gill, 2017)) ( Figure 9).…”