Date: Tuesday, April 22, 2014
Time: 11:00 AM PDT
Key topics will include:
- A brief overview on CRISPR Cas9 Technology and its applications
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CRISPR products available in different host systems
- Information and a demo of gRNA design tool
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Technologies developed to minimize off-target effects
- gRNA libraries
Learn more about CRISPR Genome Engineering and how it can benefit your research.
Abstract:
Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR, is an attractive next-generation genome-engineering tool due to its ease of use and high efficiency in a range of host systems. This exciting technology enables precise genome editing, including gene knock-outs and knock-ins using a Cas9 nuclease and targeting short guide RNA (sgRNA).
The Cas9 nuclease is targeted to specific genomic sites by the first 20 nucleotides of a gRNA, Several independent groups have shown that this system is prone to off-target mutations, even at sites that differ by as many as 5 nt from the intended target sequence. DNA2.0 has developed a series of solutions to suppress off-target effects by controlling Cas9 expression and developing a gRNA design bioinformatics tool. Furthermore, we have developed the NickaseNinja™ vector which offers high fidelity in a convenient, single-vector co-expressing two tandem sgRNAs using dual RNA polymerase promoters (patent pending). The NicakseNinja vector consists of the Cas9 nickase mutant, which utilizes double nicking of DNA to minimize off-target mutagenesis by 50-1,000 fold, as displayed in prior publications from the Dr. Feng Zhang lab at the Broad Institute (Cambridge, MA). The CRISPR NickaseNinja system facilitates targeted knock-down with efficiencies over 50%. In addition, an emerging application using sgRNA libraries with a lenti-viral delivery mechanism to provide means of large-scale, loss-of-function screens will be further discussed.
