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Nat Biotechnol. 2018 Jul;36(6):540-546. doi: 10.1038/nbt.4147. Epub 2018 May 21.

High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast.

Guo X1,2, Chavez A1,2,3, Tung A1, Chan Y1,2, Kaas C1,2,4, Yin Y5, Cecchi R1, Garnier SL1, Kelsic ED1,2, Schubert M1,2, DiCarlo JE1,2,6, Collins JJ1,7,8,9,10, Church GM1,2.

Author information

1
Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA.
2
Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
3
Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.
4
Department of Expression Technologies 2, Novo Nordisk A/S, Maaloev, Denmark.
5
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
6
Department of Ophthalmology, Columbia University College of Physicians and Surgeons, New York, New York, USA.
7
Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
8
Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
9
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
10
Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

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.

PMID:
29786095
PMCID:
PMC5990468
DOI:
10.1038/nbt.4147
[Indexed for MEDLINE]
Free PMC Article

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