A new tool to direct CRISPR to multiple targets

HIRI researchers simplify method for editing multiple DNA sections in parallel

CRISPR-Cas-based gene editing has been used for a large variety of applications. The system requires guide RNAs, whose sequence specifically determines the site where DNA or RNA modification occurs. Scientists at the Helmholtz Institute for RNA-based Infection Research (HIRI) describe a powerful means to assemble CRISPR arrays, part of CRISPR-Cas systems that encode multiple guida RNAs. Assembled CRISPR arrays can then be used to direct the gene editing machine to multiple parallel targets. The HIRI is a joint institution of the Helmholtz Centre for Infection Research (HZI) in Braunschweig and the Julius Maximilian University of Würzburg. The novel tool CRATES enabled Prof Chase Beisel and his research group “Synthetic Biology of RNA” to gain new insights into the biology of CRISPR-Cas systems.

©HIRI©HIRI The researchers at the HIRI developed a powerful means to assemble CRISPR arrays, revealing properties to guide CRISPR array design and enabling highly multiplexed targeting. Employing this new approach revealed new insights into the biology of CRISPR-Cas systems, the source of CRISPR technologies.

What is it all about?
CRISPR technologies represent powerful tools for genome editing that are revolutionizing basic research, medicine, biotechnology, and agriculture. One key feature of these technologies is their ability to multiplex—or target multiple genes at one time—by using multiple guide RNAs. One major challenge is how to best encode these guide RNAs. The exquisite solution CRISPR-Cas systems offer is in the form of CRISPR arrays. These arrays consist of identical sequence repeats alternating with unique spacers. Each repeat-spacer pair becomes a guide RNA. CRISPR arrays are highly compact and can encode hundreds of unique guide RNAs. However, these same arrays have proven incredibly difficult to construct, limiting our basic understanding of their properties and how they can be used for multiplexing applications with CRISPR technologies.

What are the key findings?
In Nature Communications, Beisel and his postdoctoral researcher Dr Chunyu Liao report a powerful means to construct individual CRISPR arrays. This approach, which they call CRATES (for CRISPR Assembly through Trimmed Ends of Spacers), allowed them to create not only long CRISPR arrays but also libraries of CRISPR arrays, enabling highly multiplexed editing or rapidly screening combinations of edits. They further used the arrays to gain new insights into the biology of CRISPR-Cas systems, working toward a more advanced understanding of this source of CRISPR technologies.

Why is this new method so powerful?
CRATES has the potential to advance the many applications of CRISPR technologies requiring multiplexing, such as editing multiple genes to simulate complex diseases, rapidly optimizing industrial bacteria for sustainable chemical production, improving CRISPR gene drives, and developing multi-input diagnostics. Furthermore, the researchers were able to derive important insights into CRISPR arrays that represent one-half of all CRISPR-Cas systems yet have been far less studied or applied.

For further reading, please see the "Behind the paper" article on nature.com

Original publication:

Chunyu Liao, Fani Ttofali, Rebecca A. Slotkowski, Steven R. Denny, Taylor D. Cecil, Ryan T. Leenay, Albert J. Keung & Chase L. Beisel: Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis. Nat Commun 2019 DOI: 10.1038/s41467-019-10747-3

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