Poster Presentations
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Single-nucleotide resolution interventions using CRISPR/Cas9
Leonidas Bleris, Professor, University of Texas at Dallas
CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. Here we demonstrate that the lentiviral-mediated CRISPR/Cas9 technology can be used to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wild-type alleles. Specifically, we used a novel gRNA-constraint-mediated method to target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9.
CRISPR-based self-cleaving mechanism for controllable gene delivery in human cells
Leonidas Bleris, Professor, University of Texas at Dallas
Controllable gene delivery via vector-based systems remains a formidable challenge in mammalian synthetic biology and a desirable asset in gene therapy applications. Here we introduce a methodology to control the copies and residence time of a gene product delivered in host human cells but also selectively disrupt fragments of the delivery vehicle. A crucial element of the proposed system is the CRISPR protein Cas9. Upon delivery, Cas9 guided by a custom RNA sequence cleaves the delivery vector at strategically placed targets thereby inactivating a co-expressed gene of interest. Importantly, using experiments in human embryonic kidney cells, we show that specific parameters of the system can be adjusted to fine-tune the delivery properties.
Fast and Sensitive Detection of Indels Induced by Precise Gene Targeting
Eric Bennett, Associate Professor, University of Copenhagen
The emerging Gene editing tools for precise engineering of higher eukaryote genomes such as ZFNs, TALENs and CRISPR/Cas have revolutionized bioscience. In contrast to the speed by which these editing tools are being optimized and strategies for high throughput use in whole-genome screens are devised, considerable less focus are being devoted to improving capabilities for detection and characterization of the induced insertions and/or deletions (indels) at the specific breakpoint as well as at potential off-targets. Currently used methods for indel identification include enzyme mismatch cleavage assays or Sanger sequencing, which do not provide sensitive, reliable and accurate indel identification and/or are costly, time and labor intensive, and poorly suited for high throughput indel screening. Here, we report a novel strategy that combines use of a simple amplicon labelling strategy with the high throughput capability of DNA fragment analysis by automated Capillary Electrophoresis for simple detection and characterization of indels induced by precise gene targeting. The strategy is coined IDAA3 for Indel Detection by Amplicon Analysis, and comparable analysis with currently used indel detection methods demonstrate that IDAA is superior and suitable for detecting indels in both cell pools with low efficiency targeting, single sorted cells and off-target evaluation.
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