MAY 10, 2018 6:00 AM PDT

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

C.E. Credits: CEU
Speaker
  • 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.


    Show Resources
    You May Also Like
    JAN 23, 2020 9:00 AM PST
    C.E. CREDITS
    JAN 23, 2020 9:00 AM PST
    DATE: January 23, 2020 TIME: 9:00am PST, 12:00pm EST...
    APR 07, 2020 8:00 AM PDT
    C.E. CREDITS
    APR 07, 2020 8:00 AM PDT
    DATE: April 7, 2020 TIME: 8:00am PT, 11:00am ET This webinar sets out to establish why quality control is key to robust, reliable, reproducible science. We will look at best practice criteri...
    MAY 08, 2020 10:00 AM PDT
    C.E. CREDITS
    MAY 08, 2020 10:00 AM PDT
    DATE: May 8, 2020 TIME: 10:00am PT, 11:00am MT, 1:00pm ET The application of next generation sequencing to interrogate immune repertoires and methods in which these highly complex dataset...
    MAR 03, 2020 9:00 AM JST
    C.E. CREDITS
    MAR 03, 2020 9:00 AM JST
    DATE: March 3, 2020 TIME: 9:00am JST A major limitation in the ex vivo expansion of harvested human hematopoietic stem-progenitor cells (HSPCs) is the rapid differentiation of HSPCs at the e...
    FEB 25, 2020 9:00 AM PST
    C.E. CREDITS
    FEB 25, 2020 9:00 AM PST
    Learn about how to generate a small scale CAR-T workflow using ThermoFisher products See detailed characterization tools that can be utilized and applied in a CAR-T workflow...
    DEC 10, 2019 9:00 AM PST
    C.E. CREDITS
    DEC 10, 2019 9:00 AM PST
    DATE: December 10, 2019TIME: 9:00am PST, 12:00pm EST A major limitation in the ex vivo expansion of harvested human hematopoietic stem-progenitor cells (HSPCs) is the rapid dif...
    Loading Comments...
    Show Resources
    Attendees
    • See more