Multiplexed precision genome editing with trackable genomic barcodes in yeast
July 1, 2018
... (Torrey Pines) for further processing. The sub-passage and culture sampling steps were triggered by a pre-defined OD (0.6), not by time elapsed. Liquid transfers were performed automatically using a Freedom EVO liquid handling system (Tecan), which was controlled by custom Pegasus software (Tecan). For the colony count analysis for survival on 5-fluoro-cytosine (5-FC) versus YPD, a strain harbori ... rol of Cas9 expression. Analysis of editing, barcoding, and plasmid removal kinetics For experiments described in Figure 2a and 2b, cells were cultured in 48-well plates in an Infinite plate reader (Tecan) at 30°C with orbital shaking. OD600 was followed by taking measurements every ~15 minutes. Cultures were maintained in log phase growth by passaging cultures every 2 doublings, when an aliquo
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.
Roy, KR; Smith, JD; Vonesch, SC; Lin, G; Tu, CS; Lederer, AR; Chu, A; Suresh, S; Nguyen, M; Horecka, J; Tripathi, A; Burnett, WT; Morgan, MA; Schulz, J; Orsley, KM; Wei, W; Aiyar, RS; Davis, RW; Bankaitis, VA; Haber, JE; Salit, ML; St Onge, RP; Steinmetz, LM;
Journal: Nat. Biotechnol. Pages: 512-520
Original article (29734294)