Genetic materials inside the nucleus are not randomly distributed but spatially organized. For example, certain genomic loci are preferentially located at the 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 <i>Saccharomyces cerevisiae</i>, “DNA zip codes” are short DNA sequences that encode spatial information targeting towards defined compartments in the nucleus. Similarly, in higher eukaryotes such as mammalian cells, DNA sequences around 5-10kb in size have been identified for spatial targeting from ~200kb bacterial artificial chromosomes (BACs) harboring human genomic sequences. However, previous strategies a have two major drawbacks: 1) the screening relies heavily on random integration of candidate DNA fragments, which suffers from no control over the copy number, no prior knowledge about the landing sites, and potential bias towards open chromatin regions; 2) the construction of BAC libraries by traditional recombineering method is labor-intensive and time-consuming. To overcome these bottlenecks, here we present a new strategy that couples the specificity of CRISPR/Cas9 system together with the large-cargo capability of serine recombinases to achieve targeted rewiring of preselected endogenous loci in mammalian cells. Moreover, direct cloning techniques are applied to quickly generate BAC library (20~100kb) with desired deletions in order 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 expand the current paradigm of large-scale genome engineering in mammalian systems, leading to novel knowledge about gene regulation on an epigenetic level.

New strategy for identification of DNA zip codes via targeted rewiring of endogenous loci in mammalian cells

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.