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Nat Biotechnol. 2018 Jul;36(6):512-520. doi: 10.1038/nbt.4137. Epub 2018 May 7.

Multiplexed precision genome editing with trackable genomic barcodes in yeast.

Author information

1
Stanford Genome Technology Center, Stanford University, Palo Alto, California, USA.
2
Genome-Scale Measurements Group, Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, USA.
3
Joint Initiative for Metrology in Biology, Stanford, California, USA.
4
Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.
5
European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.
6
Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA.
7
Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, USA.
8
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA.
9
Department of Chemistry, Texas A&M University, College Station, Texas, USA.
10
Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts, USA.

Abstract

Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here we describe a CRISPR-Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide-donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA-Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.

PMID:
29734294
PMCID:
PMC5990450
DOI:
10.1038/nbt.4137
[Indexed for MEDLINE]
Free PMC Article

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