CREEPY: CRISPR-mediated editing of synthetic episomes in yeast

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.

Automated summary

The use of synthetic genomics has revolutionized our ability to answer fundamental biological questions by designing and building "big" DNA constructs. Saccharomyces cerevisiae, or budding yeast, is a powerful platform for assembling large synthetic constructs, but introducing designer variations to episomal assemblies remains challenging. The CRISPR Engineering of EPisomes in Yeast (CREEPY) method provides a rapid and efficient way to edit large synthetic episomal DNA constructs.