Volume 94 Issue 44 | p. 13 | Concentrates
Issue Date: November 7, 2016

Protein evolution’s CRISPR way forward

The CRISPR-X technique allows for targeted mutagenesis in directed evolution studies
Department: Science & Technology
News Channels: Biological SCENE
Keywords: biotechnology, directed evolution, CRISPR, somatic hypermutation, mutagenesis

Directed evolution is a powerful tool that allows researchers to simulate natural selection. Many such experiments place human genes into bacteria or yeast. Researchers then randomly mutate those genes in hopes of improving a protein’s function. But Gaelen T. Hess and colleagues of Stanford University wanted to give directed evolution experiments more guidance and do the entire process in cultured human cells. So they turned to CRISPR gene editing for inspiration and developed a new method called CRISPR-X, which allows scientists to study effects of random mutations in small stretches of DNA (Nat. Methods 2016, DOI: 10.1038/nmeth.4038). First, a catalytically dead version of the protein Cas9, called dCas9, is directed to a location along the genome matching its guide RNA. To induce mutations in the region, the team appended two hairpin loops that bind a viral protein called MS2 to the guide RNA. Finally, the MS2 protein, which the researchers fused to a mutation-inducing enzyme called AID, is expressed in the cell. There, it is recruited to the dCas9, enabling hypermutation at that site. “This is much more targeted than what people have done before,” Hess says. He foresees researchers using the system to mutate two different protein binding sites to study protein-protein interactions and to study mammalian regulatory elements that turn genes on and off.

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Fused MS2 and AID proteins are recruited to the dCas9 site where AID induces DNA mutations (stars).
Credit: Nat. Methods
A schematic of the CRISPR-X method shows dCas9 recruiting AID for localized hypermutation.
 
Fused MS2 and AID proteins are recruited to the dCas9 site where AID induces DNA mutations (stars).
Credit: Nat. Methods
 
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ISSN 0009-2347
Copyright © American Chemical Society

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