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Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array

By Bernd Zetsche, Matthias Heidenreich, Prarthana Mohanraju, Iana Fedorova, Jeroen Kneppers, Ellen M DeGennaro, Nerges Winblad, Sourav R Choudhury, Omar O Abudayyeh, Jonathan S Gootenberg, Wen Y Wu, David A Scott, Konstantin Severinov, John van der Oost, Feng Zhang

Posted 01 Oct 2016
bioRxiv DOI: 10.1101/049122 (published DOI: 10.1038/nbt0217-178b)

Microbial CRISPR-Cas defense systems have been adapted as a platform for genome editing applications built around the RNA-guided effector nucleases, such as Cas9. We recently reported the characterization of Cpf1, the effector nuclease of a novel type V-A CRISPR system, and demonstrated that it can be adapted for genome editing in mammalian cells. Unlike Cas9, which utilizes a trans-activating crRNA (tracrRNA) as well as the endogenous RNaseIII for maturation of its dual crRNA:tracrRNA guides, guide processing of the Cpf1 system proceeds in the absence of tracrRNA or other Cas (CRISPR associated) genes, suggesting that Cpf1 is sufficient for pre-crRNA maturation. This has important implications for genome editing, as it would provide a simple route to multiplex targeting. Here, we show for two Cpf1 orthologs that no other factors are required for array processing and demonstrate multiplex gene editing in mammalian cells as well as in the mouse brain by using a designed single CRISPR array.

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