The CRISPR-Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. One of the key components of this editing system is Cas9 endonuclease. The cleavage activity of the S. pyogenes Cas9 enzyme is mediated by the coordinated functions of two catalytic domains and creates blunt-ended, double-stranded breaks. Alanine substitution at key residues within these domains creates two Cas9 nickase variants. Variant D10A produces a nick on the targeting strand, while H840A nicks the non-targeting strand. This double nicking strategy can be leveraged to reduce unwanted off-target effect. However, the nickase experiments can be inherently more complicated than standard CRISPR-Cas9 editing, given the requirement for two guide RNAs to function simultaneously.
In this webinar, both Shuqi Yan and Mollie Schubert will present the data from the characterization of a number of factors that impact the efficiency of cooperative nicking in cell cultures. They will also summarize a few key design considerations for achieving efficient gene disruption or homology directed repair (HDR) when planning your nickase experiments.
DATE:
Thursday, February 22, 2018
TIME:
9:00 am CST (UTC-6 hours)
1:00 pm CST (UTC-6 hours)
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Presenter: Shuqi Yan
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Research Scientist
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Integrated DNA Technologies
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Shuqi Yan is a research scientist in the Molecular Genetics research group at IDT. Shuqi earned her master’s degree in molecular genetics and microbiology at Stony Brook University in New York, where she studied the impact of genetic composition on gene expression in various model organisms. At IDT, she has been focusing on genome editing applications and is leading the development of nickase applications for genome editing in mammalian cells.
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Presenter: Mollie Schubert
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Research Scientist
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Integrated DNA Technologies
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Mollie Schubert is a research scientist in the Molecular Genetics research group at IDT. Mollie received her master’s degree in biochemistry from Iowa State University, and has been at IDT since 2013. Recently, she has been focusing on CRISPR genome editing systems. In particular, she has been conducting high throughput screening of CRISPR-Cas9 guides for the development of a guide RNA design tool, and also optimizing the composition and delivery of the RNP complexes in cell culture.
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