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Competitive Genomic Screens of Barcoded Yeast Libraries
Published on: August 11, 2011
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Multiplexed precision genome editing with trackable genomic barcodes in yeast.
Kevin R Roy1,2,3,4, Justin D Smith1,4, Sibylle C Vonesch5
1Stanford Genome Technology Center, Stanford University, Palo Alto, California, USA.
Nature Biotechnology
|May 8, 2018
Summary
Researchers developed MAGESTIC, a CRISPR-Cas9 tool for precise genome editing in yeast. This method enables high-throughput analysis of genotype-phenotype relationships by integrating cellular barcodes for robust tracking.
Area of Science:
- Genetics
- Molecular Biology
- Biotechnology
Background:
- Understanding genotype-phenotype links requires scalable genome alteration and phenotypic assessment.
- Current methods face limitations in precision and throughput for large-scale genetic studies.
Purpose of the Study:
- To introduce MAGESTIC (Multiplexed Accurate Genome Editing with Short, Trackable, Integrated Cellular barcodes), a novel CRISPR-Cas9 based method for yeast.
- To enable high-throughput genome editing and robust phenotyping in Saccharomyces cerevisiae.
Main Methods:
- Utilized array-synthesized guide-donor oligos for plasmid-based editing.
- Integrated genomic barcodes to prevent loss and ensure accurate phenotyping.
- Employed a LexA-Fkh1p fusion protein to enhance donor DNA recruitment and editing efficiency.
Main Results:
- Achieved over fivefold increase in editing efficiency.
- Performed saturation editing of the essential SEC14 gene, identifying critical amino acids for lipid signaling inhibition.
- Generated thousands of natural genetic variants and characterized guide mismatch tolerance.
- Identified cryptic Pol III termination elements impacting guide efficacy.
Conclusions:
- MAGESTIC offers a powerful platform for multiplexed and accurate genome editing in yeast.
- The method facilitates uncovering the genetic basis of complex phenotypes.
- Broad applicability for yeast genetic research and functional genomics.
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