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Nat Methods. 2019 Sep;16(9):887-893. doi: 10.1038/s41592-019-0508-6. Epub 2019 Aug 12.

Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts.

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Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands.
Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.
Department of Chemistry, University of Basel, Basel, Switzerland.


The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.

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