Multiplexed precision genome editing with trackable genomic barcodes in yeast View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2018-05-07

AUTHORS

Kevin R Roy, Justin D Smith, Sibylle C Vonesch, Gen Lin, Chelsea Szu Tu, Alex R Lederer, Angela Chu, Sundari Suresh, Michelle Nguyen, Joe Horecka, Ashutosh Tripathi, Wallace T Burnett, Maddison A Morgan, Julia Schulz, Kevin M Orsley, Wu Wei, Raeka S Aiyar, Ronald W Davis, Vytas A Bankaitis, James E Haber, Marc L Salit, Robert P St Onge, Lars M Steinmetz

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. More... »

PAGES

512

References to SciGraph publications

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nbt.4137

DOI

http://dx.doi.org/10.1038/nbt.4137

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1103843570

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/29734294


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