Simultaneous multi-site editing of individual genomes using retron arrays

During recent years the use of libraries-scale genomic manipulations scaffolded on CRISPR gRNAs have been transformative. However, these existing approaches are typically multiplexed across genomes. Unfortunately, building cells with multiple, non-adjacent precise mutations remains a laborious cycle of editing, isolating an edited cell, and editing again. The use of bacterial retrons can overcome this limitation. Retrons are genetic systems composed of a reverse transcriptase and a non-coding RNA (ncRNA) that contains an msd, which is reverse transcribed to produce multiple copies of single-stranded DNA. Here, we describe a technology - termed a multitron - for precisely modifying multiple sites on a single genome simultaneously using retron arrays, in which multiple donor-encoding msds are produced from a single transcript. The multitron architecture is compatible with both recombineering in prokaryotic cells and CRISPR editing in eukaryotic cells. We demonstrate applications for this approach in molecular recording, genetic element minimization, and metabolic engineering.