(56a) A New Strategy for Rapid Identification of DNA Zip Codes Via Targeted Rewiring of Endogenous Loci in Mammalian Cells

Genetic materials inside the nucleus are not randomly distributed but spatially organized. For example, certain genes are preferentially located at nuclear periphery, while others are more likely to be found at nuclear interior. However, the underlying mechanisms that regulate such spatial distribution of the genome remain largely unknown. Originally discovered in Saccharomyces cerevisiae, “DNA zip codes” are short DNA sequences that encode spatial information targeting towards defined nuclear compartments. Similarly, in higher eukaryotes such as mammalian cells, cis-regulatory DNA elements around 5-10kb in size have been identified for spatial targeting from ~220kb bacterial artificial chromosomes (BACs) harboring human genomic sequences. However, previous strategies used to identify mammalian DNA zip codes have two major drawbacks: 1) the functional screening relies heavily on the random genomic integration of candidate DNA fragments, which suffers from no control over the integration copy number, no prior knowledge about the integration landing sites, and potential bias towards open chromatin regions; 2) the construction of BAC deletion libraries by traditional recombineering method is labor-intensive and time-consuming. To overcome these bottlenecks, in this work we present a new strategy that takes advantage of the specificity of CRISPR/Cas9 system as well as the large-cargo capability of the serine recombinases to achieve targeted rewiring of preselected endogenous loci in mammalian cells. Moreover, direct cloning techniques are applied to quickly generate BACs with desired deletions to accelerate the library construction for downstream screening. Using this strategy, the targeting function of each modified BAC can be evaluated at the same, defined locus, which excludes undesired chromosomal context effects associated with random integration. Therefore, our strategy enables the dissection to be carried out in a more consistent, reliable and efficient manner. Collectively, we anticipate this strategy to greatly facilitate the mechanistic dissection of complex relationships between DNA sequences and nuclear organization, leading to novel knowledge about gene regulation on an epigenetic level.