Journal Article

Multiplexed precision genome editing with trackable genomic barcodes in yeast

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

2018

Overview

A method to introduce defined mutations into the yeast genome enables saturation mutagenesis of a gene and genome-scale introduction of genetic variants. 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.

• Publisher: Springer New York,Nature Publishing Group
• Subject: 45 / 45/41 / 45/70 / 49 / 631/1647 / 631/1647/1513/1967/3196 / 631/208 / 631/208/191/1908 / 631/553 / Agriculture / Amino Acid Substitution / Amino acids / Baking yeast / Bar codes / Bioinformatics / Biomedical Engineering/Biotechnology / Biomedicine / Biotechnology / Cloning / CRISPR / CRISPR-Cas Systems / Deoxyribonucleic acid / DNA / DNA Barcoding, Taxonomic - methods / DNA sequencing / DNA, Fungal - genetics / Editing / Efficiency / Fusion protein / Gene Editing - methods / Genetic aspects / Genetic diversity / Genetic variance / Genome editing / Genome, Fungal / Genomes / Genomics / Genotypes / Homologous Recombination / Life Sciences / Lipids / Methods / Multiplexing / Organic chemistry / Phenotypes / Phenotyping / Phospholipid Transfer Proteins - genetics / Plasmids / Plasmids - genetics / Proteins / RNA, Fungal - genetics / Saccharomyces cerevisiae / Saccharomyces cerevisiae - genetics / Saccharomyces cerevisiae Proteins - genetics / Saturation mutagenesis / Yeast / Yeasts (Fungi)