Dispersal plays a prominent role in most conceptual models of community assembly. However, direct measurement of dispersal across a whole community is difficult at ecologically relevant spatial scales. For cryptic organisms, such as fungi and bacteria, the scale and importance of dispersal limitation has become a major point of debate. We use an experimental island biogeographic approach to measure the effects of dispersal limitation on the ecological dynamics of an important group of plant symbionts, ectomycorrhizal fungi. We manipulated the isolation of uncolonized host seedlings across a natural landscape and used a range of molecular techniques to measure the dispersal rates of ectomycorrhizal propagules and host colonization. Some species were prolific dispersers, producing annual spore loads on the order of trillions of spores per km(2). However, fungal propagules reaching host seedlings decreased rapidly with increasing distance from potential spore sources, causing a concomitant reduction in ectomycorrhizal species richness, host colonization and host biomass. There were also strong differences in dispersal ability across species, which correlated well with the predictable composition of ectomycorrhizal communities associated with establishing pine forest. The use of molecular tools to measure whole community dispersal provides a direct confirmation for a key mechanism underlying island biogeography theory and has the potential to make microbial systems a model for understanding the role of dispersal in ecological theory.

CRISPR-Cas genome engineering in yeast has relied on preparation of complex expression plasmids for multiplexed gene knockouts and point mutations. Here we show that co-transformation of a single linearized plasmid with multiple PCR-generated guide RNA (gRNA) and donor DNA cassettes facilitates high-efficiency multiplexed integration of point mutations and large constructs. This technique allowed recovery of marker-less triple-engineering events with 64% efficiency without selection for expression of all gRNAs. The gRNA cassettes can be easily made by PCR and delivered in any combination. We employed this method to rapidly phenotype up to five specific allele combinations and identify synergistic effects. To prototype a pathway for the production of muconic acid, we integrated six DNA fragments totaling 24 kb across three loci in naive Saccharomyces cerevisiae in a single transformation. With minor modifications, we integrated a similar pathway in Kluyveromyces lactis. The flexibility afforded by combinatorial gRNA delivery dramatically accelerates complex strain engineering for basic research and industrial fermentation.

Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80–100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, we created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Our strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.

Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.

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5Tremendous genetic variation exists in nature, but our ability to create and characterize 6 individual genetic variants remains far more limited in scale. Likewise, engineering proteins and 7 phenotypes requires the introduction of synthetic variants, but design of variants outpaces 8 experimental measurement of variant effect. Here, we optimize efficient and continuous 9 generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded 10 DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing 11 efficiency can be obtained using this method, enabling multiplexed applications. We introduce 12 Retron Library Recombineering (RLR), a system for high-throughput screens of variants, 13 wherein the association of introduced edits with their retron elements enables a targeted deep 14 sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized 15 variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared 16 genomic DNA of an evolved bacterium, experimentally querying millions of sequences for 17 antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize 18 non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus 19 present new avenues for exploring variation, both designed and not, across the entire genome. 20 21 Introduction: 22Constructing genotypes of interest and observing their effect on phenotype critically aids 23 our understanding of genetics and genome function. As methods for editing genomes have 24 progressed, this "reverse genetics" approach has expanded in breadth and scale, from knockout 25 libraries 1 to refactored genomes 2,3 . These experiments can now be performed within multiplexed 26 pools, which allow an ever greater number of mutations to be explored across varied conditions. 27Critically, both creating genotypes and observing phenotype within pools has necessitated 28 2 development of new techniques. Transposon insertions 4 , marked integrations 5 and CRISPR-29 inhibition 6,7 can create thousands of variants simultaneously within pooled experiments, and 30 targeted sequencing of these elements enables pooled measurement of variant phenotypes. These 31 advancements in experimental scale have fundamentally transformed our understanding of 32 genome function 8 . 33However, these current high-throughput genetic techniques remain limited, in that they 34 typically introduce, ablate, or regulate kilobases of DNA to create variation. This contrasts with 35 point mutations, which are ubiquitous in natural variation 9 , and are indispensable for engineering 36 proteins 10 and metabolic pathways 11 . While modifying kilobases of DNA can add and subtract 37 functional elements such as genes and regulatory sequences from the genome, point mutations 38 can alter the function of these elements, accessing a larger phenotypic landscape. 39Point mutations and other precision edits can be performed by oligonucleo...

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We describe here the Genotype Specification Language (GSL), a language that facilitates the rapid design of large and complex DNA constructs used to engineer genomes. The GSL compiler implements a high-level language based on traditional genetic notation, as well as a set of low-level DNA manipulation primitives. The language allows facile incorporation of parts from a library of cloned DNA constructs and from the "natural" library of parts in fully sequenced and annotated genomes. GSL was designed to engage genetic engineers in their native language while providing a framework for higher level abstract tooling. To this end we define four language levels, Level 0 (literal DNA sequence) through Level 3, with increasing abstraction of part selection and construction paths. GSL targets an intermediate language based on DNA slices that translates efficiently into a wide range of final output formats, such as FASTA and GenBank, and includes formats that specify instructions and materials such as oligonucleotide primers to allow the physical construction of the GSL designs by individual strain engineers or an automated DNA assembly core facility.