ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time

Abstract

Thanks to its sophistication, the CRISPR/Cas system has been a widely used yeast genome editing method. However, CRISPR methods generally rely on preassembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose an alternative method, Assembly and CRISPR-targeted in vivo Editing (ACtivE), that only relies on in vivo assembly of linear DNA fragments for plasmid and donor DNA construction. Thus, depending on the user’s need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share, and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight loci proximal to autonomously replicating sequences (ARS) in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing a β-carotene pathway. In only a few days, >80% integration efficiency for single gene integration and >50% integration efficiency for triplex integration were achieved on Saccharomyces cerevisiae BY4741 from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.

The synergetic effect from the combination of different adsorption resins in batch and semi‐continuous cultivations of S. Cerevisiae cell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol

“…The S. cerevisiae strains used in this study were a CEN.PK2‐1C‐originated yeast strain (EUROSCARF), and a CEN.PK2‐1C yeast strain previously engineered by (Walls, Malcı, Nowrouzi, Li, d'Espaux, et al, 2021 ) labeled as LRS6 with the following genotype: MATa, leu2‐3, 112::HIS3MX6‐GAL1p‐ERG19/GAL10p‐ERG8; ura3‐52::URA3‐GAL1p‐MvaSA110G/GAL10p‐MvaE; his3Δ1::hphMX4‐GAL1p‐ERG12/GAL10p‐IDI1; trp1‐289::TRP1_GAL1p‐CrtE (X. dendrorhous)/GAL10p ERG20; YPRCdelta15::NatMX‐GAL1p‐CrtE/GAL10p‐CrtE; ARS1014::GAL1p‐TASY‐GFP; ARS1622b::GAL1p‐MBP‐TASY‐ERG20; ARS1114a::TDH3p‐MBP‐TASY‐ERG20; ARS511b::GAL1p‐T5αOH/GAL3‐CPR. Using the CRISPR Cas9 methodologies described by (Malci et al, 2022 ) Malcı et al ( 2022 ), the strain EJ2 was created with the following genotype ( LRS6 , RKC3::GAL1‐TAT).…”

The synergetic effect from the combination of different adsorption resins in batch and semi-continuous cultivations ofS. cerevisiaecell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol

“…The S. cerevisiae strains used in this study were a CEN.PK2-1C-originated yeast strain (EUROSCARF, Germany), and a CEN.PK2-1C yeast strain previously engineered by (Walls et al, 2021) labeled as LRS6 with the following genotype: MATa, leu2-3, 112::HIS3MX6-GAL1p-ERG19/GAL10p-ERG8; ura3-52::URA3-GAL1p-MvaSA110G/GAL10p-MvaE; his3Δ1::hphMX4-GAL1p-ERG12/GAL10p-IDI1; trp1-289::TRP1_GAL1p-CrtE (X. dendrorhous)/GAL10p ERG20; YPRCdelta15::NatMX-GAL1p-CrtE/GAL10p-CrtE; ARS1014::GAL1p-TASY-GFP; ARS1622b::GAL1p-MBP-TASY-ERG20; ARS1114a::TDH3p-MBP-TASY-ERG20; ARS511b::GAL1p-T5αOH/GAL3-CPR. Using the CRISPR Cas9 methodologies described by (Malci et al, 2022) Malci et al (2022), the strain EJ2 was created with the following genotype ( LRS6 , RKC3::GAL1-TAT).…”

“…Using the CRISPR Cas9 methodologies described by (Malci et al, 2022) Malci et al (2022), the strain EJ2 was created with the following genotype (LRS6, RKC3::GAL1-TAT).…”

Multiplex CRISPR-Cas Genome Editing: Next-Generation Microbial Strain Engineering

“…Currently, multiplex genome editing technology is primarily utilized to stably express heterologous genes by integrating them into the genome (Table ). Antioxidants such as astaxanthin , and β-carotene, , which can be used as food additives, are being produced in S. cerevisiae through heterologous gene expression.…”