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Title: Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica

Abstract

Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.

Authors:
 [1];  [2];  [2];  [3];  [4];  [5];  [5];  [1];  [1];  [5];  [4];  [2];  [1]
+ Show Author Affiliations
  1. Univ. of California, Riverside, CA (United States)
  2. USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
  3. United States Military Academy, West Point, NY (United States)
  4. Univ. of Texas, Austin, TX (United States)
  5. Clemson Univ., Clemson, SC (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Science Foundation (NSF)
OSTI Identifier:
1597716
Alternate Identifier(s):
OSTI ID: 1779369
Grant/Contract Number:  
AC02-05CH11231; CSP-503076; NSF 1706545; N00014-15-1-2785; NSF 1706134; NSF 1526742
Resource Type:
Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 55; Journal Issue: C; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Schwartz, Cory, Cheng, Jan-Fang, Evans, Robert, Schwartz, Christopher A., Wagner, James M., Anglin, Scott, Beitz, Adam, Pan, Weihua, Lonardi, Stefano, Blenner, Mark, Alper, Hal S., Yoshikuni, Yasuo, and Wheeldon, Ian. Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica. United States: N. p., 2019. Web. doi:10.1016/j.ymben.2019.06.007.
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Schwartz, Cory, Cheng, Jan-Fang, Evans, Robert, Schwartz, Christopher A., Wagner, James M., Anglin, Scott, Beitz, Adam, Pan, Weihua, Lonardi, Stefano, Blenner, Mark, Alper, Hal S., Yoshikuni, Yasuo, & Wheeldon, Ian. Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica. United States. https://doi.org/10.1016/j.ymben.2019.06.007
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Schwartz, Cory, Cheng, Jan-Fang, Evans, Robert, Schwartz, Christopher A., Wagner, James M., Anglin, Scott, Beitz, Adam, Pan, Weihua, Lonardi, Stefano, Blenner, Mark, Alper, Hal S., Yoshikuni, Yasuo, and Wheeldon, Ian. Sun . "Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica". United States. https://doi.org/10.1016/j.ymben.2019.06.007. https://www.osti.gov/servlets/purl/1597716.
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@article{osti_1597716,
title = {Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica},
author = {Schwartz, Cory and Cheng, Jan-Fang and Evans, Robert and Schwartz, Christopher A. and Wagner, James M. and Anglin, Scott and Beitz, Adam and Pan, Weihua and Lonardi, Stefano and Blenner, Mark and Alper, Hal S. and Yoshikuni, Yasuo and Wheeldon, Ian},
abstractNote = {Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.},
doi = {10.1016/j.ymben.2019.06.007},
journal = {Metabolic Engineering},
number = C,
volume = 55,
place = {United States},
year = {Sun Jun 16 00:00:00 EDT 2019},
month = {Sun Jun 16 00:00:00 EDT 2019}
}
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Publisher's Version of Record
https://doi.org/10.1016/j.ymben.2019.06.007
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    Works referencing / citing this record:

    A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity
    journal, January 2020

    Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica
    journal, November 2019

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    • Microbial Cell Factories, Vol. 18, Issue 1
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    Stress-tolerant non-conventional microbes enable next-generation chemical biosynthesis
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    • Thorwall, Sarah; Schwartz, Cory; Chartron, Justin W.
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    A set of Yarrowia lipolytica CRISPR/Cas9 vectors for exploiting wild-type strain diversity
    journal, January 2020

    Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica
    journal, November 2019

    • Ganesan, Vijaydev; Spagnuolo, Michael; Agrawal, Ayushi
    • Microbial Cell Factories, Vol. 18, Issue 1
    • DOI: 10.1186/s12934-019-1259-x
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