A complex landscape of genomic regulatory elements underpins patterns of metazoan gene expression, yet it has been technically difficult to disentangle composite regulatory elements within their endogenous genomic context. Expression of the Sox2 transcription factor (TF) in mouse embryonic stem cells (mESCs) depends on a distal regulatory cluster of DNase I hypersensitive sites (DHSs), but the contributions of individual DHSs and their degree of independence remain a mystery. Here, we comprehensively analyze the regulatory architecture of the Sox2 locus in mESCs using Big-IN to scarlessly deliver payloads ranging up to 143 kb, permitting deletions, rearrangements and inversions of single or multiple DHSs, and surgical alterations to individual TF recognition sequences. Multiple independent mESC clones were derived for each payload, extensively sequence-verified, and profiled for expression of Sox2 specifically from the engineered allele. We find that a single core DHS comprising a handful of key TF recognition sequences is sufficient to sustain significant expression in mESCs, though its contribution is modulated by additional DHSs. Moreover, their overall activity is influenced by specific DHS order and/or orientation effects. We built a highly predictive model for locus regulation which includes nonlinear components indicating both synergy and redundancy among. Our results suggest that composite regulatory elements and their influence on gene expression can be resolved to a tractable set of sequence features using synthetic regulatory genomics.

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The Sc2.0 project is building a eukaryotic synthetic genome from scratch, incorporating thousands of designer features. A major milestone has been achieved with the assembly of all individual Sc2.0 chromosomes. Here, we describe the consolidation of multiple synthetic chromosomes using endoreduplication intercross to generate a strain with 6.5 synthetic chromosomes. Genome-wide chromosome conformation capture and long-read direct RNA sequencing were performed on this strain to evaluate the effects of designer modifications, such as loxPsym site insertion, tRNA relocation, and intron deletion, on 3D chromosome organization and transcript isoform profiles. To precisely map "bugs", we developed a method, CRISPR Directed Biallelic URA3-assisted Genome Scan, or CRISPR D-BUGS, exploiting directed mitotic recombination in heterozygous diploids. Using this method, we first fine-mapped a synII defect resulting from two loxPsym sites in the 3′ UTR of SHM1. This approach was also used to map a combinatorial bug associated with synIII and synX, revealing a highly unexpected genetic interaction that links transcriptional regulation, inositol metabolism and tRNASerCGA abundance. "Starvation" for tRNASerCGA leads to insufficient levels of the key positive inositol biosynthesis regulator, Swi3, which contains tandem UCG codons. Finally, to further expedite consolidation, we employed a new method, chromosome swapping, to incorporate the largest chromosome (synIV), thereby consolidating more than half of the Sc2.0 genome in a single strain.

Use of synthetic genomics to design and build ″big″ DNA has revolutionized our ability to answer fundamental biological questions by employing a bottom-up approach. S. cerevisiae, or budding yeast, has become the major platform to assemble large synthetic constructs thanks to its powerful homologous recombination machinery and the availability of well-established molecular biology techniques. However, efficiently and precisely introducing designer variations to episomal assemblies remains challenging. Here, we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, for rapid engineering of mammalian DNA constructs larger than 100 kb. We demonstrate that editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes with CRISPR. After optimizing CREEPY for episomal editing, we achieve efficient simplex and multiplex editing as demonstrated by engineering a mouse Sox2-harboring episome.

Use of synthetic genomics to design and build ‘big’ DNA has revolutionized our ability to answer fundamental biological questions by employing a bottom-up approach. Saccharomyces cerevisiae, or budding yeast, has become the major platform to assemble large synthetic constructs thanks to its powerful homologous recombination machinery and the availability of well-established molecular biology techniques. However, introducing designer variations to episomal assemblies with high efficiency and fidelity remains challenging. Here we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, a method for rapid engineering of large synthetic episomal DNA constructs. We demonstrate that CRISPR editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes. We optimize CREEPY for efficient and precise multiplex editing of >100 kb yeast episomes, providing an expanded toolkit for synthetic genomics.