High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR–Cas9 in yeast
Name
nihms961343.pdf
Description
Accepted version
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806.15 KB
Format
Adobe PDF
Checksum (MD5)
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Author(s)
Collins, James J.
Date Issued
May 21, 2018
Journal
Nature biotechnology
Publisher
Springer Science and Business Media LLC
Citation
Guo, Xiaoge et al. “High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR–Cas9 in yeast.” Nature biotechnology 36 (2018) © 2018 The Author(s)
Version
Author's final manuscript
Abstract
Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80-100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, we created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Our strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.
Subjects
Biotechnology
Molecular Medicine
Applied Microbiology and Biotechnology
Bioengineering
Biomedical Engineering
MIT Department
Massachusetts Institute of Technology. Institute for Medical Engineering & Science
Massachusetts Institute of Technology. Department of Biological Engineering
Terms of Use
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1038/nbt.4147
