JAN 26, 2021 8:00 AM PST

High-throughput CRISPR editing using the Onyx™ platform identifies essential residues in proteins

Sponsored by: Inscripta
C.E. Credits: P.A.C.E. CE Florida CE
  • Postdoctoral Research Fellow
      Liselot Dewachter studies fundamental bacterial physiology in the hopes of finding new ways to kill bacteria, including antibiotic-resistant species. She obtained her PhD from the University of Leuven in Belgium where she worked in Prof. Jan Michiels' lab, which focuses on antibiotic tolerance and the development of new antimicrobial therapies. Toward the end of her PhD, she started focusing on essential genes in the bacterium Escherichia coli that can be used as targets for new antibiotics. In collaboration with Inscripta, she uses high-throughput CRISPR editing to map essential amino acid residues in the genes of interest. After finishing her PhD, Dr. Dewachter moved to Switzerland where she is now working as a postdoc in Prof. Jan-Willem Veening's lab. She is still harnessing the power of CRISPR in a genome-wide CRISPRi screen designed to identify important genes in the pathogen Streptococcus pneumoniae.
    • Scientific Liaison, EMEA, Inscripta
        Laura Klitten has worked with scientific communication within medical genetics, non-coding RNAs, molecular biology and new technologies in a variety of industrial roles for the last 8 years. Dr. Klitten is the Scientific Liaison with Inscripta's EMEA team. Dr. Klitten is, among other tasks, responsible for correct and timely communication between internal and external partners and act as the "voice of customer" within Inscripta to ensure outstanding support and execution of projects demonstrating the many applications of the Onyx Digital Genome Engineering platform. Before joining Inscripta, Dr. Klitten had different commercial roles including Global Product Manager at QIAGEN and Exiqon. Dr. Klitten earned her M.Sc. and Ph.D degrees at the University of Copenhagen, Denmark.

      Date:  January 26, 2021
      Time: 8:00am (PST),  11:00am (EST)
      Over the past years, CRISPR/Cas genome editing has quickly become the method of choice for genetic manipulation, owing to its ease-of-use, flexibility, and efficiency. However, another main advantage of CRISPR technology is its scalability. In the study presented during this webinar, we used the high-throughput CRISPR-based Onyx platform to perform saturation mutagenesis on four different essential genes involved in cell envelope synthesis in E. coli. In total, 22,790 edits were designed so that every amino acid of these proteins would be replaced by every other amino acid. We used these saturation mutagenesis libraries to probe the essentiality of all different residues. Edits that could not be introduced point to mutations that do not support the viability and, hence, residues essential for protein function. This way, we were able to identify known essential amino acids, i.e. catalytic residues and residues involved in substrate binding, thereby validating our experimental approach. Additionally, studying the Onyx libraries' composition also pinpointed several residues that were previously not known to be essential. Besides offering vastly improved insights into protein function, we expect our results to help fight against antibiotic resistance. By providing insight into essentiality at an amino-acid level, we expect to aid structure-based drug design targeted against these essential proteins.
      Learning Objectives
      • Describe how to quickly perform saturation mutagenesis in E.coli using the high-throughput CRISPR-based ONYX platform
      • Report how to probe for residues essential for protein function
      • Explore the flexibility in amino acid tolerance
      Webinars will be available for unlimited on-demand viewing after live event.
      LabRoots is approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E. ® Program. By attending this webinar, you can earn 1 Continuing Education credit once you have viewed the webinar in its entirety.

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