MAY 10, 2018 06:00 AM PDT

Multiplexed Precision Genome Editing with Trackable Genome-Integrated Barcodes in Yeast

C.E. CREDITS: CEU | P.A.C.E. CE | Florida CE
Speakers
  • Postdoctoral Scholar, Department of Genetics, Stanford University
    Biography
      Kevin obtained his Ph.D. at UCLA in the laboratory of Guillaume Chanfreau, where he studied RNA biology and developed high-throughput sequencing methods to map RNA degradation intermediates genome-wide. He is currently a National Research Council (NRC) postdoctoral associate in the laboratories of Dr. Lars Steinmetz at Stanford University and Dr. Marc Salit at the the Joint Initiative for Metrology in Biology (JIMB), a joint institute between Stanford University and the National Institute of Standards and Technology (NIST). Kevin works on developing high-throughput precision genome editing technologies with CRISPR/Cas9 to enable dissecting the genetic architecture underlying complex cellular phenotypes.

    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. In this presentation I will highlight a CRISPR/Cas9-based method in S. cerevisiae for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC). MAGESTIC uses array-synthesized oligonucleotides encoding guide RNA-donor DNA pairs with a sophisticated cloning strategy for plasmid-based high-throughput editing. By linearizing the guide-donor plasmid in vivo concomitant with integration at a genomic barcode locus, MAGESTIC circumvents problems associated with post-editing plasmid barcode loss and enables robust phenotyping with one-to-one barcode-to-cell correspondence. We demonstrate that editing efficiency can be increased >5-fold by actively recruiting donor DNA directly to the site of breaks using the LexA-Fkh1p fusion protein. As a proof of principle, 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 in yeast. MAGESTIC will create opportunities to unravel the genetic basis of quantitative traits, map functional residues on proteins and RNAs across entire pathways, dissect DNA regulatory elements, and build improved organisms for biotechnology.


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