Multiplex engineering of industrial yeast genomes using CRISPRm.
Global demand has driven the use of industrial strains of the yeast Saccharomyces cerevisiae for large-scale production of biofuels and renewable chemicals. However, the genetic basis of desired domestication traits is poorly understood because robust genetic tools do not exist for industrial hosts. We present an efficient, marker-free, high-throughput, and multiplexed genome editing platform for industrial strains of S. cerevisiae that uses plasmid-based expression of the CRISPR/Cas9 endonuclease and multiple ribozyme-protected single guide RNAs. With this multiplex CRISPR (CRISPRm) system, it is possible to integrate DNA libraries into the chromosome for evolution experiments, and to engineer multiple loci simultaneously. The CRISPRm tools should therefore find use in many higher-order synthetic biology applications to accelerate improvements in industrial microorganisms.
['Ryan|Owen W|OW|', 'Cate|Jamie H D|JH|']
D001483:Base Sequence D064130:CRISPR-Associated Proteins D064113:CRISPR-Cas Systems D064112:Clustered Regularly Interspaced Short Palindromic Repeats D019020:Directed Molecular Evolution D004720:Endonucleases D015723:Gene Library D005818:Genetic Engineering D016681:Genome, Fungal D007218:Industrial Microbiology D008969:Molecular Sequence Data D010957:Plasmids D017394:RNA, Guide D012441:Saccharomyces cerevisiae
Industrial yeast CRISPRm Saccharomyces cerevisiae Multiplex genome editing Cas9 DNA libraries Synthetic biology Chromosomal integration Directed evolution Homology-directed repair