A synthetic RNA-mediated evolution system in yeast

Abstract

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.

Targeting Transcriptional and Translational Hindrances in a Modular T7RNAP Expression System in Engineered Pseudomonas putida

“…For comparison, the wild-type TΦ terminator has a TE of approximately 79% . The tZ terminator has already been successfully applied to prevent read-through by several research groups. − We used the newly constructed pT7-eGFP-tZ plasmid to generate DNA fragments for a new set of T7 RNA polymerase transcription assays in the same fashion as described above for the pT7-eGFP plasmid with the TΦ terminator. In this case, however, the DNA fragment “tZ” (containing the T7 promoter, eGFP and the tZ terminator only) is the fragment which, after transcription, yields the reference length mRNA transcript.…”

Recent Advances in Directed Yeast Genome Evolution

“…CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) [ 71 ], in vivo continuous evolution (ICE) [ 72 ] and eukaryotic multiplex automated genome engineering (eMAGE) [ 73 ] normally accelerated the evolution process of specific proteins or pathways instead of the whole genome in yeast. However, when employing these methods to evolve transcriptional factors, it is highly possible to cause diverse phenotypes.…”

Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts

“…Oligos DAM1-4 and DAM6 were used individually with DAM594 to amplify pCfB3050 making up plasmids pDAM1-4 and pDAM6. pDAM7 and pDAM8 were made by amplifying the gRNA cassettes from pDAM1 and pDAM2 with‚ oligos TJOS-62 and TJOS-65 and assembled into pEDJ400 and pEDJ437 39 , respectively. Oligos DAM218 + DAM594 and DAM219 + DAM594 amplified pDAM1 to give plasmids pDAM77 and pDAM78, DAM485 + DAM594 amplified pDAM8 to make pDAM182, and DAM335 + DAM594 amplified pDAM7 to make pDAM146.…”

“…Strain CEN.PK2-1C and BY4741 (EUROSCARF) were transformed with pEDJ391 39 to express Cas9 (CPK1 and SBY1). About 1–2 μg for each plasmid or fragment of DNA was used in all chemical transformations of S. cerevisiae .…”