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
    DEC 02, 2020 8:00 AM PST
    Add to Calendar Select one of the following: iCal Google Calendar Outlook Calendar Yahoo Calendar
    C.E. CREDITS
    DEC 02, 2020 8:00 AM PST
    Add to Calendar Select one of the following: iCal Google Calendar Outlook Calendar Yahoo Calendar
    DATE: December 2nd, 2020 TIME: 08:00am PDT, 11:00pm EDT Bioreactors and shakers are used to cultivate microorganisms, plant, insect, and mammalian cells in different volumes. Upscaling of pr...
    OCT 08, 2020 7:00 AM PDT
    C.E. CREDITS
    OCT 08, 2020 7:00 AM PDT
    DATE: October 8, 2020 TIME: 7:00am PDT, 10:00am EDT, 4:00pm CEST How often do you pipette in your cell culture lab every day? Usually, we do it so often that we tend stop thinking about ho...
    NOV 16, 2020 8:00 AM PST
    C.E. CREDITS
    NOV 16, 2020 8:00 AM PST
    Date: November 16, 2020 Time: 8:00am (PST), 11:00am (EST) CRISPR screening has become the prime discovery tool in modern biomedical research and drug discovery. At the same time, most screen...
    OCT 29, 2020 6:00 AM PDT
    C.E. CREDITS
    OCT 29, 2020 6:00 AM PDT
    Date: October 29, 2020 Time: 6:00am (PDT), 9:00am (EDT), Chronic inflammation can occur as a result of a combination of genetic predispositions and environmental factors. Epigenetic modifica...
    SEP 10, 2020 9:00 AM PDT
    C.E. CREDITS
    SEP 10, 2020 9:00 AM PDT
    Date: September 10, 2020 Time: 9:00am (PDT), 12:00pm (EDT) Osmolality testing is relevant throughout the entire bioprocessing workflow. As customers look to refine mAb and gene therapy workf...
    SEP 02, 2020 7:00 AM PDT
    C.E. CREDITS
    SEP 02, 2020 7:00 AM PDT
    DATE: September 2, 2020 TIME: 03:00pm PDT, 6:00pm EDT Spatial omics is an expanding collection of methods to examine biological molecules in their geographical context. By retaining the prec...
    Loading Comments...
    Show Resources
    Attendees
    • See more