A synthetic RNA-mediated evolution system in yeast.

Emil D Jensen,Jay D Keasling,Michael K Jensen,Marcos Laloux,Beata J Lehka,Lasse E Pedersen,Tadas Jakočiūnas

doi:10.1093/nar/gkab472

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.

Highlights

The ability to evolve biomolecules with tailor-made properties is inherently linked to mutagenesis, driving both natural and laboratory evolution
In order to develop a targeted in vivo evolution system, we initially sought to combine elements of RNA-programmed genome targetability of CRISPR/Cas9, and error-prone RNA polymerase for expression of donor-coupled chimeric gRNAs, serving as repair templates at targeted genomic loci [31,32,34]
Beyond orthogonal transcription relying on the high T7 promoterspecificity and synthesis of untranslated RNA in yeast by T7RNAP [37], transcriptional mutagenesis can be adjusted by evolved T7RNAPs with nucleotide substitution error rates up to 1.25×10–3 demonstrated in vitro and in E. coli [38], making T7RNAP of particular interest for in vivo evolution

Summary

Introduction

The ability to evolve biomolecules with tailor-made properties is inherently linked to mutagenesis, driving both natural and laboratory evolution. While the vast majority of these systems rely on targeted mutagenesis of genomic loci using variant DNA donors designed and generated in vitro [4,5,6,7], a number of evolution systems have been developed to couple mutation and selection cycles in vivo in both bacteria [2,8,9,10,11], yeast [12,13,14,15], and mammalian cells [16] Such strategies circumvent the need for repeated cycles of human-guided design of mutational spectra, tedious hands-on genetic library construction, transformation, and selection, and have enabled targeted per-base substitution rates >10 000-fold higher than those of host genomes (e.g. 10–5–10–4 per base) [14,17,18]. It has been demonstrated that RNA molecules synthesized in vivo can confer genome editing following induced DSBs [28,29]

Methods

Results

Conclusion