Dataset Information

Easy efficient HDR-based targeted knock-in in Saccharomyces cerevisiae genome using CRISPR-Cas9 system.

ABSTRACT: During the last two decades, yeast has been used as a biological tool to produce various small molecules, biofuels, etc., using an inexpensive bioprocess. The application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein (Cas) techniques in yeast genetic and metabolic engineering has made a paradigm shift, particularly with a significant improvement in targeted chromosomal integration using synthetic donor constructs, which was previously a challenge. This study reports the CRISPR-Cas9-based highly efficient strategy for targeted chromosomal integration and in-frame expression of a foreign gene in the genome of Saccharomyces cerevisiae (S. cerevisiae) by homology-dependent recombination (HDR); our optimized methods show that CRISPR-Cas9-based chromosomal targeted integration of small constructs at multiple target sites of the yeast genome can be achieved with an efficiency of 74%. Our study also suggests that 15 bp microhomology flanked arms are sufficient for 50% targeted knock-in at minimal knock-in construct concentration. Whole-genome sequencing confirmed that there is no off-target effect. This study provides a comprehensive and streamlined protocol that will support the targeted integration of essential genes into the yeast genome for synthetic biology and other industrial purposes.Highlights• CRISPR-Cas9 based in-frame expression of foreign protein in Saccharomyces cerevisiae using Homology arm without a promoter.• As low as 15 base pairs of microhomology (HDR) are sufficient for targeted integration in Saccharomyces cerevisiae.• The methodology is highly efficient and very specific as no off-targeted effects were shown by the whole-genome sequence.

SUBMITTER: Singh R

PROVIDER: S-EPMC10109214 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Easy efficient HDR-based targeted knock-in in <i>Saccharomyces cerevisiae</i> genome using CRISPR-Cas9 system.

Singh Rajveer R Chandel Shivani S Ghosh Arijit A Gautam Anupam A Huson Daniel H DH Ravichandiran V V Ghosh Dipanjan D

Bioengineered 20220601 6

During the last two decades, yeast has been used as a biological tool to produce various small molecules, biofuels, etc., using an inexpensive bioprocess. The application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein (Cas) techniques in yeast genetic and metabolic engineering has made a paradigm shift, particularly with a significant improvement in targeted chromosomal integration using synthetic donor constructs, which was previously a challenge ...[more]

PMID: 36602175

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Project description:UnlabelledThe clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3'→5' exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters.ImportanceRecent improvements in cloning strategies for biosynthetic gene clusters promise rapid advances in understanding and exploiting natural products in the environment. For manipulation of such biosynthetic gene clusters to generate valuable bioactive compounds, efficient and specific gene editing of these large DNA fragments is required. In this study, a highly efficient in vitro DNA editing system has been established. When combined with end repair using T4 DNA polymerase, Cas9 precisely and seamlessly catalyzes targeted editing, including in-frame deletion or insertion of the gene(s) of interest. This in vitro CRISPR editing (ICE) system promises a step forward in our ability to engineer biosynthetic pathways.

Project description:BackgroundThe abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers.ResultsThe aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L- 1 after 48 h under oxygen limited condition in bioreactor fermentations.ConclusionThis work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast's expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Dataset Information

Easy efficient HDR-based targeted knock-in in Saccharomyces cerevisiae genome using CRISPR-Cas9 system.

Publications

Easy efficient HDR-based targeted knock-in in <i>Saccharomyces cerevisiae</i> genome using CRISPR-Cas9 system.

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