Targeted In Vivo Mutagenesis in Yeast Using CRISPR/Cas9 and Hyperactive Cytidine and Adenine Deaminases.

Directed evolution is a preferred strategy to improve the function of proteins such as enzymes that act as bottlenecks in metabolic pathways. Common directed evolution approaches rely on error-prone PCR-based libraries where the number of possible variants is usually limited by cellular transformation efficiencies. Targeted in vivo mutagenesis can advance directed evolution approaches and help to overcome limitations in library generation. In the current study, we aimed to develop a high-efficiency time-controllable targeted mutagenesis toolkit in the yeast Saccharomyces cerevisiae by employing the CRISPR/Cas9 technology. To that end, we fused the dCas9 protein with hyperactive variants of adenine and cytidine deaminases aiming to create an inducible CRISPR-based mutagenesis tool targeting a specific DNA sequence in vivo with extended editing windows and high mutagenesis efficiency. We also investigated the effect of guide RNA multiplexing on the mutagenesis efficiency both phenotypically and on the DNA level.

Multiplexed Guide RNA Expression Leads to Increased Mutation Frequency in Targeted Window Using a CRISPR-Guided Error-Prone DNA Polymerase in Saccharomyces cerevisiae.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, with its ability to target a specific DNA locus using guide RNAs (gRNAs), is particularly suited for targeted mutagenesis. The targeted diversification of nucleotides in Saccharomyces cerevisiae using a CRISPR-guided error-prone DNA polymerase─called yEvolvR─was recently reported. Here, we investigate the effect of multiplexed expression of gRNAs flanking a short stretch of DNA on reversion and mutation frequencies using yEvolvR. Phenotypic assays demonstrate that higher reversion frequencies are observed when expressing multiple gRNAs simultaneously. Next generation sequencing reveals a synergistic effect of multiple gRNAs on mutation frequencies, which is more pronounced in a mutant with a partially defective DNA mismatch repair system. Additionally, we characterize a galactose-inducible yEvolvR, which enables temporal control of mutagenesis. This study demonstrates that multiplex expression of gRNAs and induction of mutagenesis greatly improves the capabilities of yEvolvR for generation of genetic libraries in vivo.

Controlling gene expression with deep generative design of regulatory DNA.

Design of de novo synthetic regulatory DNA is a promising avenue to control gene expression in biotechnology and medicine. Using mutagenesis typically requires screening sizable random DNA libraries, which limits the designs to span merely a short section of the promoter and restricts their control of gene expression. Here, we prototype a deep learning strategy based on generative adversarial networks (GAN) by learning directly from genomic and transcriptomic data. Our ExpressionGAN can traverse the entire regulatory sequence-expression landscape in a gene-specific manner, generating regulatory DNA with prespecified target mRNA levels spanning the whole gene regulatory structure including coding and adjacent non-coding regions. Despite high sequence divergence from natural DNA, in vivo measurements show that 57% of the highly-expressed synthetic sequences surpass the expression levels of highly-expressed natural controls. This demonstrates the applicability and relevance of deep generative design to expand our knowledge and control of gene expression regulation in any desired organism, condition or tissue.

Fluorescence-Activated Cell Sorting as a Tool for Recombinant Strain Screening.

Metabolic engineering of microbial cells is the discipline of optimizing microbial metabolism to enable and improve the production of target molecules ranging from biofuels and chemical building blocks to high-value pharmaceuticals. The advances in genetic engineering have eased the construction of highly engineered microbial strains and the generation of genetic libraries. Intracellular metabolite-responsive biosensors facilitate high-throughput screening of these libraries by connecting the levels of a metabolite of interest to a fluorescence output. Fluorescent-activated cell sorting (FACS) enables the isolation of highly fluorescent single cells and thus genotypes that produce higher levels of the metabolite of interest. Here, we describe a high-throughput screening method for recombinant yeast strain screening based on intracellular biosensors and FACS.

Expansion of the Yeast Modular Cloning Toolkit for CRISPR-Based Applications, Genomic Integrations and Combinatorial Libraries.

Standardisation of genetic parts has become a topic of increasing interest over the last decades. The promise of simplifying molecular cloning procedures, while at the same time making them more predictable and reproducible has led to the design of several biological standards, one of which is modular cloning (MoClo). The Yeast MoClo toolkit provides a large library of characterised genetic parts combined with a comprehensive and flexible assembly strategy. Here we aimed to (1) simplify the adoption of the standard by providing a simple design tool for including new parts in the MoClo library, (2) characterise the toolkit further by demonstrating the impact of a BglII site in promoter parts on protein expression, and (3) expand the toolkit to enable efficient construction of gRNA arrays, marker-less integration cassettes and combinatorial libraries. These additions make the toolkit more applicable for common engineering tasks and will further promote its adoption in the yeast biological engineering community.

Does co-expression of Yarrowia lipolytica genes encoding Yas1p, Yas2p and Yas3p make a potential alkane-responsive biosensor in Saccharomyces cerevisiae?

Alkane-based biofuels are desirable to produce at a commercial scale as these have properties similar to current petroleum-derived transportation fuels. Rationally engineering microorganisms to produce a desirable compound, such as alkanes, is, however, challenging. Metabolic engineers are therefore increasingly implementing evolutionary engineering approaches combined with high-throughput screening tools, including metabolite biosensors, to identify productive cells. Engineering Saccharomyces cerevisiae to produce alkanes could be facilitated by using an alkane-responsive biosensor, which can potentially be developed from the native alkane-sensing system in Yarrowia lipolytica, a well-known alkane-assimilating yeast. This putative alkane-sensing system is, at least, based on three different transcription factors (TFs) named Yas1p, Yas2p and Yas3p. Although this system is not fully elucidated in Y. lipolytica, we were interested in evaluating the possibility of translating this system into an alkane-responsive biosensor in S. cerevisiae. We evaluated the alkane-sensing system in S. cerevisiae by developing one sensor based on the native Y. lipolytica ALK1 promoter and one sensor based on the native S. cerevisiae CYC1 promoter. In both systems, we found that the TFs Yas1p, Yas2p and Yas3p do not seem to act in the same way as these have been reported to do in their native host. Additional analysis of the TFs suggests that more knowledge regarding their mechanism is needed before a potential alkane-responsive sensor based on the Y. lipolytica system can be established in S. cerevisiae.

Model-Assisted Fine-Tuning of Central Carbon Metabolism in Yeast through dCas9-Based Regulation.

Engineering Saccharomyces cerevisiae for industrial-scale production of valuable chemicals involves extensive modulation of its metabolism. Here, we identified novel gene expression fine-tuning set-ups to enhance endogenous metabolic fluxes toward increasing levels of acetyl-CoA and malonyl-CoA. dCas9-based transcriptional regulation was combined together with a malonyl-CoA responsive intracellular biosensor to select for beneficial set-ups. The candidate genes for screening were predicted using a genome-scale metabolic model, and a gRNA library targeting a total of 168 selected genes was designed. After multiple rounds of fluorescence-activated cell sorting and library sequencing, the gRNAs that were functional and increased flux toward malonyl-CoA were assessed for their efficiency to enhance 3-hydroxypropionic acid (3-HP) production. 3-HP production was significantly improved upon fine-tuning genes involved in providing malonyl-CoA precursors, cofactor supply, as well as chromatin remodeling.

Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation Using Csy4 in Saccharomyces cerevisiae.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has greatly accelerated the field of strain engineering. However, insufficient efforts have been made toward developing robust multiplexing tools in Saccharomyces cerevisiae. Here, we exploit the RNA processing capacity of the bacterial endoribonuclease Csy4 from Pseudomonas aeruginosa, to generate multiple gRNAs from a single transcript for genome editing and gene interference applications in S. cerevisiae. In regards to genome editing, we performed a quadruple deletion of FAA1, FAA4, POX1 and TES1 reaching 96% efficiency out of 24 colonies tested. Then, we used this system to efficiently transcriptionally regulate the three genes, OLE1, HMG1 and ACS1. Thus, we demonstrate that multiplexed genome editing and gene regulation can be performed in a fast and effective manner using Csy4.