https://video.igem.org/videos/watch/0a7eef3a-76f2-4a06-b632-4f8da8f1fe79 Title: SHUFFLE: Self-iterative High-throughput Unlocked Flexible Frequency Loci Editor Description: Improving microbial cell factories requires precise, diverse mutations across multiple loci, yet conventional genome-editing methods are limited by insufficient targeting control, restricted substitution spectra, or double-strand-break toxicity. We developed SHUFFLE (Self-iterative High-throughput Unlocked Flexible Frequency Loci Editor), a targeted N-to-N base-editing platform combining dual-activity TadDE, Cas9 nickase, and paired guide RNAs. Iterative engineering expanded PAM recognition with nSpCas9-NG, incorporated AlkA and CWBEmini modules, and evolved TadDE using EvolvePro to obtain A158R. The optimized TadDE_A158R–nSpCas9_NG–AlkA editor achieved up to approximately 50% editing efficiency at tested loci and generated diverse nucleotide substitutions. SHUFFLE enabled diversification of global regulators to identify strains with improved growth and polyhydroxybutyrate production on ethanol, and editing of the accA ribosome-binding site to improve malonyl-CoA availability, as indicated by flaviolin screening. SHUFFLE provides a versatile approach to targeted diversification for microbial strain engineering. Wiki | Official Page Thu, 29 Oct 2026 21:22:07 GMT https://validator.w3.org/feed/docs/rss2.html PeerTube - https://video.igem.org https://video.igem.org/client/assets/images/icons/icon-1500x1500.png https://video.igem.org/videos/watch/0a7eef3a-76f2-4a06-b632-4f8da8f1fe79 All rights reserved, unless otherwise specified in the terms specified at https://video.igem.org/about and potential licenses granted by each content's rightholder.