De novoassembly, delivery and expression of a 101 kb human gene in mouse cells

Abstract

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a pipeline for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kb. The DNA assembly step is supported by an integrated robotic workcell. We assembled the 101 kb human HPRT1 gene in yeast, delivered it to mouse embryonic stem cells, and showed expression of the human protein from its full-length gene. This pipeline provides a framework for producing systematic, designer variants of any mammalian gene locus for functional evaluation in cells. Significance StatementMammalian genomes consist of a tiny proportion of relatively well-characterized coding regions and vast swaths of poorly characterized "dark matter" containing critical but much less well-defined regulatory sequences. Given the dominant role of noncoding DNA in common human diseases and traits, the interconnectivity of regulatory elements, and the importance of genomic context, de novo design, assembly, and delivery can enable large-scale manipulation of these elements on a locus scale. Here we outline a pipeline for de novo assembly, delivery and expression of mammalian genes replete with native regulatory sequences. We expect this pipeline will be useful for dissecting the function of non-coding sequence variation in mammalian genomes.

Big DNA as a tool to dissect an age-related macular degeneration-associated haplotype

“…2D-E). 30 Several dozen clones with the correct structure by junction PCR were fully sequence confirmed by whole-genome sequencing of the yeast strain (Fig. 2F).…”

Big DNA as a tool to dissect an age-related macular degeneration-associated haplotype

“…We identified complete assemblies by amplifying across the assembly junctions in an automated RT-PCR workflow (Fig. 2D-E) (30). Several dozen clones with the correct structure by junction PCR were fully sequence confirmed by whole-genome sequencing of the yeast strain (Fig.…”

“…We are now applying these methods towards assembly of large mammalian loci and pathways called "assemblons" because they are assembled by HR in the yeast cell. These assemblons are built by a specific instantiation of our homologous recombination-based assembly method called extrachromosomal SWAP-In (eSWAP-In) that we recently described (30). Importantly, these assemblons can be designed such that they allow combinatorial assembly of one or multiple variant segments interspersed with wild-type sequence segments.…”

“…It would be simple to prepare ten variants each bearing a single risk SNV, and 40 additional variants bearing all possible combinations of two SNVs. These can then be delivered to a suitable test cell, such as a retinal pigment epithelial (RPE) cell homozygous for the protective haplotype, replacing one of those "wild-type" alleles with a landing pad similar to that described by (30). This would allow the 50 engineered loci to replace one of the endogenous alleles in a single step.…”

“…Initial screening for assembly clones was performed using a high-throughput automated RT-PCR workflow described previously (30). Briefly, 96 colonies arising from the assembly transformation were selected and grown in liquid culture before isolating genomic DNA (also containing the assemblon YAC DNA).…”