Randomizing the human genome by engineering recombination between repeat elements

Abstract While protein-coding genes are characterized increasingly well, 99% of the human genome is non-coding and poorly understood. This gap is due to a lack of tools for engineering variants that affect sequence to the necessary extent. To bridge this gap, we have developed a toolbox to create deletions, inversions, translocations, and extrachromosomal circular DNA at scale by highly multiplexed insertion of recombinase recognition sites into repetitive sequences with CRISPR prime editing. Using this strategy, we derived stable human cell lines with several thousand clonal insertions, the highest number of novel sequences inserted into single human genomes. Subsequent recombinase induction generated an average of more than one hundred megabase-sized rearrangements per cell, and thousands across the whole population. The ability to detect rearrangements as they are generated and to track their abundance over time allowed us to measure the selection pressures acting on different types of structural changes. We observed a consolidation towards shorter variants that preferentially delete growth-inhibiting genes and a depletion of translocations. We isolated and characterized 21 clones with multiple recombinase-induced rearrangements. These included viable haploid clones with deletions that span hundreds of kilobases as well as triploid HEK293T clones with aneuploidies and fold back chromosomes. We mapped the impact of these genetic changes on gene expression to decipher how structural variants affect gene regulation. The genome scrambling strategy developed here makes it possible to delete megabases of sequence, move sequences between and within chromosomes, and implant regulatory elements into new contexts which will shed light on the genome organization principles of humans and other species.