Large-scale interrogation of genome structure is crucial for understanding how genomic organization influences cellular function, yet existing methods are limited by the low density of achievable modifications or the toxicity of methods. Here we address this gap by presenting a versatile approach that combines gene editing and recombinase technologies. The protocol serves two critical purposes: (1) facilitating the introduction of hundreds to thousands of precise genomic edits per cell and (2) enabling the creation of a controlled platform to systematically investigate the effects of induced genomic rearrangements. Specifically, the method leverages prime editing to insert recombinase recognition sites (for example, loxP) into repetitive genomic regions, such as LINE-1 elements, thereby enabling extensive genetic modifications in human cells. This scale of genome editing has not previously been attainable and supports a wide range of studies, including genome-wide functional analyses and essentiality mapping. Inducing controlled rearrangements with recombinase and tracking cell survival under selective conditions allows direct mapping of genome architecture to cellular fitness, opening new opportunities for genome-wide functional screens and rational synthetic genome design. Unlike methods that rely on double-strand breaks or random transposon insertion, this Protocol supports a programmable installation of thousands of recombination sites at repeat elements, offering denser and more predictable substrates for controlled genome rearrangement. The full protocol takes ~12–18 weeks to complete and requires intermediate to advanced expertise in genome editing, mammalian cell culture and sequencing analysis. This large-scale genome structure interrogation approach uses prime editing to insert recombinase recognition sites into repetitive genomic regions. It enables thousands of precise genomic edits to be installed per cell with systematic investigation of their effects.
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