Fig. 1 Major technologies for genome evolution in Saccharomyces cerevisiae, including random genome evolution and trackable genome evolutiongTME—global transcription machinery engineering; SCRaMbLE—synthetic chromosome recombination and modification by LoxP-mediated evolution; YOGE—yeast oligo-mediated genome engineering; eMAGE—eukaryotic multiplex automated genome engineering; RAGE—RNAi-assisted genome evolution; CHAnGE—CRISPR/Cas9- and homology-directed-repair-assisted genome-scale engineering; MAGIC—multi-functional genome-wide CRISPR system; MAGESTIC—multiplexed accurate genome editing with short, trackable, integrated cellular barcodes; NGS—next-generation sequencing

Fig. 2 Scheme of automated RNAi-assisted genome evolution in yeast(a) RNAi mechanism in yeast. The double-stranded RNA (dsRNA) is digested by the endonuclease Dicer into short interference RNA (siRNA), which binds to the effector protein Argonaute to form the RNA-induced silencing complex (RISC). The non-sense strand of siRNA binds to the target mRNA, leading to the degradation and interference of the transcription of the target gene. (b) Construction of a genome-wide modulation part library in the yeast strain with the reconstituted RNAi machinery. Full-length cDNA library was directionally cloned under the control of a constitutive promoter. The sense and anti-sense configurations resulted in genetic overexpression and knockdown, respectively. (c) RNAi-assisted multiplex genomic mutations in yeast. Gene modulation parts were flanked by homologous arms for iterative and multiplex δ integration into the repetitive genomic sequences. To enable efficient and selection-free δ integration, a Cas9 expression cassette was integrated into the RNAi harboring yeast strain

Fig. 3 MAGIC for genome-wide mapping genotype-phenotype relationships[34,42](a) Development of CRISPR-AID using three orthogonal CRISPR proteins, a nuclease-deficient CRISPR protein fused with an activation domain (dLbCpf1-VP) for CRISPRa, a nuclease-deficient mutant fused with a repression domain (dSpCas9-RD1152) for CRISPRi, and a catalytically active CRISPR protein (SaCas9) for CRISPRd. (b) Guide sequences for genome-wide activation (orange), interference (light blue), and deletion (magenta) were synthesized as arrayed oligos on DNA chip and cloned into the corresponding gRNA expression plasmids. The transformation of the pooled plasmid libraries into the CRISPR-AID integrated yeast strain resulted in the construction of the MAGIC library. The MAGIC library was subject to growth enrichment or high throughput screening, and the corresponding enrichment or depletion of guide sequences were profiled using next-generation sequencing. MAGIC can be employed to better understand and engineer complex phenotypes

Fig. 4 Construction of a recombinant yeast strain for simultaneous utilization of lignocellulosic carbons, such as cellobiose, xylose, and acetateXR—xylose reductase; XDH—xylitol dehydrogenase; ACS—acetyl-CoA synthetase; AADH—acetylating acetaldehyde dehydrogenase; ADH—alcohol dehydrogenase

Fig. 5 Construction of yeast cell factories for an efficient production of FFA[48](In wild-type yeast, the major carbon metabolism is to drive ethanol fermentation from glucose. In the fatty acid-overproducing yeast, metabolic engineering strategies were employed to establish efficient biosynthetic pathway from glucose to fatty acid. In the lipogenesis yeast, genome evolution was performed to completely reprogram the cellular metabolism from alcohol fermentation to fatty acid biosynthesis)PPP—pentose phosphate pathway; TCA—tricarboxylic acid; FFA—free fatty acid