Iterative recombinase technologies for efficient and precise genome engineering across kilobase to megabase scales

Genome editing technologies face challenges in achieving precise, large-scale DNA manipulations in higher organisms, including inefficiency, limited editing scales and types, and the retention of undesired sequences such as recombination sites (“scars”). Here, we present programmable chromosome engineering (PCE) and RePCE, two programmable chromosome editing systems enabling scarless kilobase-to-megabase DNA manipulations in plants and human cells. Through high-throughput engineering, we obtained Lox sites with a 10-fold reduced reversibility and applied an AI-assisted recombinase engineering method (AiCE_rec) to generate Cre variants with 3.5 times the recombination efficiency of the wild type. Incorporation of a Re-pegRNA-mediated scar-free strategy further enhanced editing precision, allowing scarless insertions, deletions, replacements, inversions, and translocations at the chromosomal level. Key applications include a 315-kb inversion in rice conferring herbicide resistance, scarless chromosome fusions, and a 12-Mb inversion at human disease-related sites. These advances significantly broaden the scope of genome editing applications in molecular breeding, therapeutic development, and synthetic biology.