Multiplexed Precision Genome Editing with Trackable Genome-Integrated Barcodes in Yeast скачать в хорошем качестве

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

Presented At: LabRoots Genetics & Genomics Virtual Event 2018 Presented By: Kevin Roy, PhD - Postdoctoral Scholar, Department of Genetics, Stanford University Speaker 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. Webinar: Multiplexed Precision Genome Editing with Trackable Genome-Integrated Barcodes in Yeast Webinar 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. Earn PACE/CME Credits: 1. Make sure you’re a registered member of LabRoots (https://www.labroots.com/virtual-even...) 2. Watch the webinar on YouTube above or on the LabRoots Website (https://www.labroots.com/virtual-even...) 3. Click Here to get your PACE (Expiration date – May 10, 2020 06:00 AM)– https://www.labroots.com/credit/pace-... 4. Click here to get your CME credits – https://www.surveymonkey.com/r/BTZQT7C LabRoots on Social: Facebook:   / labrootsinc   Twitter:   / labroots   LinkedIn:   / labroots   Instagram:   / labrootsinc   Pinterest:   / labroots   SnapChat: labroots_inc