Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision

Abstract

We developed a CRISPR-Cas9- and homology-directed-repair-assisted genome-scale engineering method named CHAnGE that can rapidly output tens of thousands of specific genetic variants in yeast. More than 98% of target sequences were efficiently edited with an average frequency of 82%. We validate the single-nucleotide resolution genome-editing capability of this technology by creating a genome-wide gene disruption collection and apply our method to improve tolerance to growth inhibitors.

Deep scanning lysine metabolism in Escherichia coli

“…While successful for phenotype optimization in industrial strains, off‐target mutations can decrease overall cell fitness and lead to “dead‐end” phenotypes, preventing further improvement of the evolved strain (Lee & Kim, ). New tools (Garst et al , ; Bao et al , ; Guo et al , ; Roy et al , ; Sadhu et al , ) that combine targeted deep scanning mutagenesis with genotype–phenotype mapping provide a powerful framework to explore distinct hypotheses in parallel, uncovering mechanisms that would be difficult to rationalize in complex systems. This concept was evident for the DapF mutations investigated here, in which lower kinetics counterintuitively improved lysine accumulation in the strains.…”

Whole‐Genome Regulation for Yeast Metabolic Engineering

“…For genome‐scale editing, it is necessary to introduce the gRNA and the corresponding DNA donor into the same cell when CRISPR system is present. Similar to the CREATE method reported in bacteria, a CRISPR/Cas9 and HDR‐assisted genome‐scale engineering (CHAnGE) method was developed by Bao et al ( Figure ) . The CRISPR guide sequence and the HDR donor were synthesized in a single oligonucleotide to facilitate the precise and efficient genome editing .…”

“…Similar to the CREATE method reported in bacteria, a CRISPR/Cas9 and HDR‐assisted genome‐scale engineering (CHAnGE) method was developed by Bao et al ( Figure ) . The CRISPR guide sequence and the HDR donor were synthesized in a single oligonucleotide to facilitate the precise and efficient genome editing . A gRNA cassette library, which included 24 765 unique sequences to target about 97.8% ORFs (6469 ORFs) annotated in budding yeast, was designed, synthesized, and assembled into a receptor plasmid.…”

“…Most recently, another CRISPR–Cas9‐based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) was developed in S. cerevisiae ( Figure ) . Instead of plasmid barcodes mentioned above, a short barcode (31‐mer) is introduced to tag each synthesized guide–donor pair during cloning in MAGESTIC. Multiple barcodes may map to the same guide–donor pair, offering internal replicates for a given edit and can serve as single‐cell tracers.…”

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing

“…To date, generating these deletion collections was a laborious process limiting their applications to a handful of strains, but several methods have been developed for genome‐scale engineering in a variety of species (e.g., Si et al, ). CRISPR‐Cas9 gene editing has been combined with genetic barcoding to enable efficient, traceable, markerless, and genome‐scale editing (Bao et al, ; Guo et al, ; Roy et al, ). For example, Bao et al () were able to use their CRISPR‐Cas‐based method combined with barcoding to identify a yeast with a 20‐fold increase the tolerance to lactic acid.…”