The DOI matches its Crossref record; 40 of the 40 references it cites were found in a registry or an index.
Selection of chromosomal DNA libraries using a multiplex CRISPR system
Open accessAbstract
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.
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Cites
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Self-citationHigh-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
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“A custom Perl script (Supplementary file 3) was written to identify all GG dinucleotide sequences in the S288C reference genome, extract every Cas9 target sequence (i.e., 20 nt sequence corresponding to the 20 nucleotides immediately 5′ of the ‘NGG’ PAM site), and obtain the genome coordinates ranging from 30 nucleoti…”
BackgroundInfluentialEthanol Can Contribute to Energy and Environmental Goals
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Cited by
The new frontier of genome engineering with CRISPR-Cas9
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Applications of CRISPR technologies in research and beyond
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A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly
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Self-citationRedox-based reagents for chemoselective methionine bioconjugation
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Genome-Editing Technologies: Principles and Applications
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Metadata
- Authors
- Ryan, O. W., Skerker, J. M., Maurer, M. J., Li, X., Tsai, J. C., Poddar, S., Lee, M. E., DeLoache, W., Dueber, J. E., Arkin, A. P., & Cate, J. H.
- Venue
- eLife
- Publisher
- eLife Sciences Publications, Ltd
- Published
- 2014-08-19
- Volume
- 3
- ISSN
- 2050-084X (ISSN-L)
- OpenAlex
- W2105689851
- Cited by
- 373
- References
- 40
- FWCI
- 11.80
- Type
- journal article
- Language
- en
- Open access
- gold
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- Ryan, O. W., Skerker, J. M., Maurer, M. J., Li, X., Tsai, J. C., Poddar, S., Lee, M. E., DeLoache, W., Dueber, J. E., Arkin, A. P., & Cate, J. H. (2014). Selection of chromosomal DNA libraries using a multiplex CRISPR system. eLife. https://doi.org/10.7554/elife.03703
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