RT Journal Article SR Electronic T1 A synthetic RNA-mediated evolution system in yeast JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.02.27.433199 DO 10.1101/2021.02.27.433199 A1 Jensen, Emil D. A1 Laloux, Marcos A1 Lehka, Beata J. A1 Pedersen, Lasse E. A1 Jakočiūnas, Tadas A1 Jensen, Michael K. A1 Keasling, Jay D. YR 2021 UL http://biorxiv.org/content/early/2021/02/28/2021.02.27.433199.abstract AB Laboratory evolution is a powerful approach to search for genetic adaptations to new or improved phenotypes, yet either relies on labour-intensive human-guided iterative rounds of mutagenesis and selection, or prolonged adaptation regimes based on naturally evolving cell populations. Here we present CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) of genomic loci using evolving chimeric donor gRNAs continuously delivered from an error-prone T7 RNA polymerase, and directly introduced as RNA repair donors into genomic targets under either Cas9 or dCas9 guidance. We validate CRAIDE by evolving novel functional variants of an auxotrophic marker gene, and by conferring resistance to a toxic amino acid analogue in baker’s yeast Saccharomyces cerevisiae with a mutation rate >3,000-fold higher compared to spontaneous native rate, thus enabling the first demonstrations of in vivo delivery and information transfer from long evolving RNA donor templates into genomic context without the use of in vitro supplied and pre-programmed repair donors.Competing Interest StatementJay D. Keasling has a financial interest in Amyris, Lygos, Demetrix, Constructive Biology, Maple Bio, and Napigen.