Engineering the RNA-guided Cascade complex for genome editing

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For humans, it took thousands of generations to develop modern domesticated plants and animals. Since the time of Thomas Morgan, scientists have been seeking methods to directly manipulate genomes of different organisms and fix faulty human genes. Precise modifications of complex genomes have been one of the major goals for scientists working in basic and applied science. Molecular tools that are currently used for genome engineering include zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). A major disadvantage of these protein-guided genome engineering systems is that the correction of each new target sequence requires laborious efforts to design a new nuclease that specifically recognizes this target sequence. Therefore, new technologies that are affordable, easy to use and efficient are still needed for the genetic engineering.***       Recently, the microbial CRISPR/Cas system has rapidly emerged as an RNA-guided genome editing tool for robust and multiplexable genome engineering. In the CRISPR mechanism, short CRISPR RNAs (crRNAs) guide the CRISPR-associated protein complexes (Cascade) or nucleases (Cas9) to complementary DNA or RNA sequences promoting specific target binding and cleavage. Successful applications of several Cas9 nucleases for genome editing and regulation have already been demonstrated in bacteria, yeast, plants, and human cells. However, the application of the Cascade complex for genome editing remains to be explored. The long-term objective of this research is to understand the molecular mechanisms of activity of Cascade and to design novel RNA-guided systems for efficient genome editing. Using the purified Cascade proteins and their complexes, we will characterize their interaction with synthetic crRNAs and target DNAs. For sequence-specific DNA cleavage and genome editing, we will introduce a nuclease domain into the engineered Cascade complex. These studies will help to optimize the protein and RNA components of engineered Cascade complexes to facilitate their delivery and assembly in targeted cells. The specificity of target recognition and cleavage by engineered Cascade complexes will be characterized in vivo using Escherichia coli and yeast cells as model systems. Our work will promote the development of novel RNA-guided molecular tools for applications in genome editing, gene therapy and biotechnology. **