Selection of chromosomal DNA libraries using a multiplex CRISPR system

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function. DOI: http://dx.doi.org/10.7554/eLife.03703.001

• Publication year

  2014

• Venue

  eLife

• Publication date

  2014-08-19

• Fields of study

  Biology, Medicine, Engineering

• Identifiers
  DOI 10.7554/eLife.03703PMID 25139909PMCID 4161972
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

• DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

  2014influential reference
• Robust and accurate prediction of residue–residue interactions across protein interfaces using evolutionary information

  2014cited by this paper
• Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation

  2014cited by this paper
• Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells

  2014cited by this paper
• Multiplex Genome Engineering Using CRISPR/Cas Systems

  2013cited by this paper
• Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay

  2013cited by this paper
• Yeast oligo-mediated genome engineering (YOGE).

  2013influential reference
• High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

  2013influential reference
• RNA-Guided Human Genome Engineering via Cas9

  2013cited by this paper
• Cas9 as a versatile tool for engineering biology

  2013cited by this paper
• DNA targeting specificity of RNA-guided Cas9 nucleases

  2013influential reference
• Crystal structure of a glucose/H+ symporter and its mechanism of action

  2013influential reference
• Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems

  2013influential reference
• Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae.

  2012cited by this paper
• Crystal structure of a bacterial homologue of glucose transporters GLUT1–4

  2012influential reference
• A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

  2012cited by this paper
• A System for the Continuous Directed Evolution of Biomolecules

  2011cited by this paper
• Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

  2011cited by this paper
• Transformation of Saccharomyces cerevisiae and other fungi

  2010cited by this paper
• Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation

  2010cited by this paper
• Cellodextrin Transport in Yeast for Improved Biofuel Production

  2010cited by this paper
• Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

  2009cited by this paper
• Enzymatic assembly of DNA molecules up to several hundred kilobases

  2009cited by this paper
• The many pathways of RNA degradation.

  2009cited by this paper
• Directed evolution drives the next generation of biocatalysts.

  2009cited by this paper
• Widespread Occurrence of Self-Cleaving Ribozymes

  2009influential reference
• Genomics of cellulosic biofuels

  2008cited by this paper
• Exporting RNA from the nucleus to the cytoplasm

  2007cited by this paper
• Structural roles of monovalent cations in the HDV ribozyme.

  2007influential reference
• The RNA polymerase III-dependent family of genes in hemiascomycetes: comparative RNomics, decoding strategies, transcription and evolutionary implications

  2006cited by this paper
• Material for : Ethanol Can Contribute To Energy and Environmental Goals

  2006influential reference
• RF cloning: a restriction-free method for inserting target genes into plasmids.

  2006cited by this paper
• What Can Replace Cheap Oil--and When?

  2005influential reference
• MUSCLE: multiple sequence alignment with high accuracy and high throughput.

  2004cited by this paper
• Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast

  2003cited by this paper
• Functional profiling of the Saccharomyces cerevisiae genome

  2002cited by this paper
• Directed evolution of a genetic circuit

  2002cited by this paper
• Identification of yeast strains using the polymerase chain reaction

  1993cited by this paper
• Analysis of the nourseothricin-resistance gene (nat) of Streptomyces noursei.

  1988cited by this paper
• A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance

  1984cited by this paper
• Bmc Molecular Biology Identification, Characterization and Expression of Novel Sex Hormone Binding Globulin Alternative First Exons in the Human Prostate

  year unknowncited by this paper

• The spliceosome assembles on excised linear introns to protect them from degradation

  2026cites this paper
• Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli

  2025cites this paper
• Sequencing of a dairy isolate unlocks Kluyveromyces marxianus as a host for lactose valorization

  2025cites this paper
• Kingdom-wide CRISPR guide design with ALLEGRO

  2025cites this paper
• RNA Polymerase III Promoters Compatible with CRISPR Gene Regulation in Saccharomyces cerevisiae

  2025cites this paper
• Advanced research and exploration of CRISPR technology in the field of directed evolution.

  2025cites this paper
• Precise measurement of molecular phenotypes with barcode-based CRISPRi systems

  2025cites this paper
• Decoding genetic diversity through genome engineering in bryophytes.

  2025cites this paper
• Recent advances in CRISPR-Cas system for Saccharomyces cerevisiae engineering.

  2025cites this paper
• A Multilayered Biocontainment System for Laboratory and Probiotic Yeast.

  2025cites this paper
• Benchmarking the PAM compatibility of Cas12a variants for high-throughput yeast genetic variant engineering

  2025cites this paper
• Unravelling fungal genome editing revolution: pathological and biotechnological application aspects

  2025cites this paper
• When good guides go bad: empirical evaluation of all unique Cas9 protospacers in E. coli reveal widespread functionality and rules for gRNA design

  2025cites this paper
• LOBSTERS: a modular vector series with a diverse set of Cas nucleases and plasmid selection markers for multiplex genome editing in Saccharomyces cerevisiae

  2025cites this paper
• Exploring CRISPR/Cas9-Mediated Gene Editing Advances in Conventional and Non-conventional Yeast Species

  2025influential citation
• Cryo-EM structure of the β-1,3-glucan synthase FKS1-Rho1 complex

  2025cites this paper
• Histone H3 cysteine 110 enhances iron metabolism and modulates replicative life span in Saccharomyces cerevisiae

  2025cites this paper
• Translation Inhibition by Rocaglamide A Enhances Susceptibility of Yeasts to Caspofungin

  2025cites this paper
• CRISPR-Cas9 genome editing of miso and soy source yeast Zygosaccharomyces sp.

  2025cites this paper
• An experimental target-based platform in yeast for screening Plasmodium vivax deoxyhypusine synthase inhibitors

  2024cites this paper
• Effect of coenzyme Q deficiency on ethanol fermentation in sake yeast.

  2024cites this paper
• The role of histone H3 leucine 126 in fine-tuning the copper reductase activity of nucleosomes

  2024cites this paper
• Further Evidence for Strong Nonneutrality of Yeast Synonymous Mutations

  2024cites this paper
• Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools.

  2024cites this paper
• Precise measurement of molecular phenotypes with barcode-based CRISPRi systems

  2024cites this paper
• Effect of S-adenosyl-methionine accumulation on hineka odor in sake brewed with a non-Kyokai yeast.

  2024cites this paper
• A unique mechanism of snRNP core assembly

  2024cites this paper
• Tuning interdomain conjugation to enable in situ population modification in yeasts

  2024cites this paper
• Multiplex CRISPR-Cas Genome Editing: Next-Generation Microbial Strain Engineering

  2024cites this paper
• “Cell Disk” DNA Storage System Capable of Random Reading and Rewriting

  2024cites this paper
• Improved gene editing and fluorescent-protein tagging in Aspergillus nidulans using a Golden Gate-based CRISPR-Cas9 plasmid system

  2024cites this paper
• Acetamidase gene as a new plasmid marker for CRISPR modification of industrial and laboratory Saccharomyces yeasts

  2024cites this paper
• Combining Oligo Pools and Golden Gate Cloning to Create Protein Variant Libraries or Guide RNA Libraries for CRISPR Applications.

  2024cites this paper
• Genome editing tools based improved applications in macrofungi

  2024cites this paper
• gUMI-BEAR, a modular, unsupervised population barcoding method to track variants and evolution at high resolution

  2023cites this paper
• Hybrid adaptation is hampered by Haldane’s sieve

  2023influential citation
• Engineering xylose fermentation in an industrial yeast: continuous cultivation as a tool for selecting improved strains.

  2023cites this paper
• Context-dependent neocentromere activity in synthetic yeast chromosome VIII

  2023cites this paper
• Deep mutational scanning highlights a role for cytosolic regions in Hrd1 function.

  2023cites this paper
• An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

  2023cites this paper
• Automation protocol for high-efficiency and high-quality genomic DNA extraction from Saccharomyces cerevisiae

  2023cites this paper
• Pathway elucidation and microbial synthesis of proaporphine and bis-benzylisoquinoline alkaloids from sacred lotus (Nelumbo nucifera)

  2023cites this paper
• Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing in Trypanosoma and Leishmania

  2023cites this paper
• CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration

  2023cites this paper
• Evaluation of Long-Term Fermentation Performance with Engineered Saccharomyces cerevisiae Strains

  2023cites this paper
• Screening non-conventional yeasts for acid tolerance and engineering Pichia occidentalis for production of muconic acid

  2023cites this paper
• A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts

  2023influential citation
• De novo production of 24-epi-ergosterol in bioengineered yeast.

  2023cites this paper
• The genomic landscape of transposable elements in yeast hybrids is shaped by structural variation and genotype-specific modulation of transposition rate

  2023cites this paper
• Targeted Mutations Produce Divergent Characteristics in Pedigreed Sake Yeast Strains

  2023cites this paper
• Rapid, scalable, combinatorial genome engineering by marker-less enrichment and recombination of genetically engineered loci in yeast

  2023cites this paper
• CRISPR genetic toolkits of classical food microorganisms: Current state and future prospects.

  2023cites this paper
• Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9

  2023cites this paper
• PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in Saccharomyces cerevisiae

  2023cites this paper
• Deep mutational scanning highlights a new role for cytosolic regions in Hrd1 function

  2023influential citation
• Sequence and chromatin features guide DNA double-strand break resection initiation.

  2023cites this paper
• Tuning Interdomain Conjugation Toward in situ Population Modification in Yeast

  2023cites this paper
• CRISPR/dCas9-Mediated Gene Silencing in Two Plant Fungal Pathogens

  2023cites this paper
• Acceleration of CRISPR/Cas9-Mediated Editing at Multiple Sites in the Saccharomyces cerevisiae Genome

  2023influential citation
• CREEPY: CRISPR-mediated editing of synthetic episomes in yeast

  2023cites this paper
• Zinc is Essential for the Copper Reductase Activity of Yeast Nucleosomes

  2023cites this paper
• Elucidation and engineering mitochondrial respiratory-related genes for improving bioethanol production at high temperature in Saccharomyces cerevisiae

  2023cites this paper
• Practical Approaches for the Yeast Saccharomyces cerevisiae Genome Modification

  2023cites this paper
• Screening non-conventional yeasts for organic acid tolerance and engineering Pichia occidentalis for production of cis,cis-muconic acid

  2023cites this paper
• Multiplex genome editing to construct cellulase engineered Saccharomyces cerevisiae for ethanol production from cellulosic biomass

  2023cites this paper
• Engineering and Evolution of the Complete Reductive Glycine Pathway in Saccharomyces cerevisiae for Formate and CO2 Assimilation

  2023cites this paper
• Polygenic Analysis of Tolerance to Carbon Dioxide Inhibition of Isoamyl Acetate “Banana” Flavor Production in Yeast Reveals MDS3 as Major Causative Gene

  2022cites this paper
• A new platform host for strong expression under GAL promoters without inducer in Saccharomyces cerevisiae

  2022cites this paper
• Biosynthesis of tetrahydropapaverine and semisynthesis of papaverine in yeast

  2022cites this paper
• On the fitness effects and disease relevance of synonymous mutations

  2022cites this paper
• Employment of the CRISPR/Cas9 system to improve cellulase production in Trichoderma reesei.

  2022cites this paper
• ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time

  2022cites this paper
• Standardization of Synthetic Biology Tools and Assembly Methods for Saccharomyces cerevisiae and Emerging Yeast Species

  2022cites this paper
• Utilizing RNA origami scaffolds in Saccharomyces cerevisiae for dCas9-mediated transcriptional control

  2022cites this paper
• Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae

  2022influential citation
• Multiple molecular events underlie stochastic switching between 2 heritable cell states in fungi

  2022cites this paper
• Rational engineering of industrial S. cerevisiae: towards xylitol production from sugarcane straw

  2022cites this paper
• Deep Mutational Scanning of Protein-Protein Interactions Between Partners Expressed from Their Endogenous Loci In Vivo.

  2022cites this paper
• Saccharomyces cerevisiae as a Heterologous Host for Natural Products.

  2022cites this paper
• Variable penetrance of Nab3 granule accumulation quantified by a new tool for high-throughput single-cell granule analysis

  2022cites this paper
• Development and expansion of the CRISPR/Cas9 toolboxes for powerful genome engineering in yeast.

  2022cites this paper
• A CRISPR-Cas9 System-Mediated Genetic Disruption and Multi-fragment Assembly in Starmerella bombicola.

  2022cites this paper
• High throughput mutagenesis and screening for yeast engineering

  2022cites this paper
• Easy efficient HDR-based targeted knock-in in Saccharomyces cerevisiae genome using CRISPR-Cas9 system

  2022cites this paper
• Unexpected growth of a classic yeast auxotroph

  2022cites this paper
• Modulating DNA Repair Pathways to Diversify Genomic Alterations in Saccharomyces cerevisiae

  2022cites this paper
• Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform

  2022cites this paper
• Nonconventional Yeasts Engineered Using the CRISPR-Cas System as Emerging Microbial Cell Factories

  2022cites this paper
• A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9

  2022influential citation
• Machine-learning guided elucidation of contribution of individual steps in the mevalonate pathway and construction of a yeast platform strain for terpenoid production.

  2022cites this paper
• Engineering Xylose Fermentation in an Industrial Yeast: Continuous Cultivation as a Tool for Selecting Improved Strains

  2022cites this paper
• Episomal editing of synthetic constructs in yeast using CRISPR

  2022cites this paper
• Advances in CRISPR/Cas9

  2022cites this paper
• RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

  2022cites this paper
• Engineering and functional analysis of yeast with a monotypic 40S ribosome subunit

  2022cites this paper
• Construction and evaluation of gRNA arrays for multiplex CRISPR‐Cas9

  2022cites this paper
• Genome Editing of Eukarya

  2021cites this paper
• Multiplex Genome Editing in Yeast by CRISPR/Cas9 – A Potent and Agile Tool to Reconstruct Complex Metabolic Pathways

  2021cites this paper
• Broadening the toolkit for quantitatively evaluating noncanonical amino acid incorporation in yeast

  2021cites this paper
• Synthetic biology is essential to unlock commercial biofuel production through hyper lipid-producing microalgae: a review

  2021cites this paper