CRISPR-Cas Assisted Shotgun Mutagenesis Method for Evolutionary Genome Engineering

Genome mutagenesis drives the evolution of organisms. Here, we developed a C RISPR-Cas a ssisted r andom m utation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgunlike manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof of principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce β-carotene. After seven rounds of iterative evolution over two months, a β-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in β-carotene production and 857 diverse genomic mutations that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes of strain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve industrially relevan S. cerevisiae CEN.PK2-1C for S-adenosyl-L-methionine production, which was increased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypes that could further be investigated by reverse engineering.