OCT 10, 2019 6:00 AM PDT

Highly-efficient and -specific genome editing with Cas9 nickase ribonucleoproteins in Primary T-cells

Sponsored by: MilliporeSigma
C.E. Credits: P.A.C.E. CE Florida CE
Speaker
  • R&D Scientist, Genome and Epigenome Editing: MilliporeSigma
    Biography
      Jacob has been working on developing and testing Cas9 products in the Genome Engineering team at MilliporeSigma since joining the company in 2015. He has led the R&D testing and has evaluated dozens of CRISPR systems, on top of helping Quality Control testing of the CRISPR products that MilliporeSigma currently sells. Recently he has focused on the development of multiple Cas9 Ribonucleoproteins and their potential therapeutic applications. Before joining MilliporeSigma, Jacob worked as a lab technician and research assistant in the Brickner Lab at Northwestern University

    Abstract

    In this webinar, we will be discussing some of our most recent testing using our Cas9 proteins, Cas9 RNP nickases in a variety of human cell types, including primary T-cells. Cas nucleases when delivered in protein format, pre-complexed with a single, modified guide RNA (gRNA), rather than encoded on a plasmid, allow researchers to bypass the historically limiting factors of using CRISPR in the therapeutic realm of off-targeting effects, low efficiency, and undesired cellular responses to foreign DNA. Modifying that Cas9 nuclease into a nickase with only a single active cutting domain, and then delivering two with proximal guide RNAs in a pair allows precise double stranded breaks, and provides hyper-specific and efficient genome editing in cell types that are more difficult to edit in.

    CRISPR/Cas technologies have unlocked vast genome editing possibilities in human cell lines but have been historically limited in the therapeutic realm by off-targeting effects, low efficiency, and undesired cellular responses to foreign DNA plasmids. Recent advancements to combat these limitations include the direct delivery of ribonucleoproteins (RNPs) to cells. In this method, Cas nucleases are delivered in protein format, pre-complexed with a single, modified guide RNA (gRNA), rather than encoded on a plasmid. Another recent advancement is the use of paired Cas9 nickase systems. A nickase is a modified Cas9 nuclease with only one active cutting domain, allowing it to create single-stranded nicks in its target DNA. To achieve a double-stranded break (DSB), nickases must utilize two individual gRNAs targeting proximal DNA sequences, making an off-target DSB nearly impossible. Here, we combine these two innovations, enabling highly-efficient editing in human cells, without observable off-target effects, via the use of paired-nickase Cas9 RNPs. The improvements this system makes on previous methods lends to greater exploration of CRISPR technologies in the therapeutic domain

    Learning Objectives:

    1. Advantages of RNPs in Primary Cells

    2. Nickase Design

    3. Therapeutic Implications of this design


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    OCT 10, 2019 6:00 AM PDT

    Highly-efficient and -specific genome editing with Cas9 nickase ribonucleoproteins in Primary T-cells

    Sponsored by: MilliporeSigma
    C.E. Credits: P.A.C.E. CE Florida CE

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    Cell Biology

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