Both CRISPR/Cas‐based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana

Friedrich Fauser; Simon Schiml; Holger Puchta

doi:10.1111/tpj.12554

journal article Jun 17, 2014

Both CRISPR/Cas‐based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana

Friedrich Fauser Simon Schiml Holger Puchta

The Plant Journal Vol. 79 No. 2 pp. 348-359 · Wiley

View at Publisher Save 10.1111/tpj.12554

Abstract

SummaryEngineered nucleases can be used to induce site‐specific double‐strand breaks (DSBs) in plant genomes. Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error‐prone non‐homologous end‐joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ. Cas9 can also be engineered to work as a nickase inducing single‐strand breaks (SSBs). Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ‐mediated mutagenesis in plants. We demonstrate the stable inheritance of nuclease‐induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 2.5 up to 70.0%. Deep sequencing analysis revealed NHEJ‐mediated DSB repair in about a third of all reads in T1 plants. In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740‐fold. Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I–SceI. Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB‐inducing enzymes. Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants. It should not only be applicable for HR‐mediated gene targeting systems but also by the combined action of two nickases as DSB‐inducing agents excluding off‐target effects in homologous genomic regions.

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685

Citations

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References

Details

Published: Jun 17, 2014
Vol/Issue: 79(2)
Pages: 348-359
License: View

Authors

F

Friedrich Fauser

Botanical Institute II Karlsruhe Institute of Technology POB 6980 76049 Karlsruhe Germany

S

Simon Schiml

Botanical Institute II Karlsruhe Institute of Technology POB 6980 76049 Karlsruhe Germany

H

Holger Puchta

Botanical Institute II Karlsruhe Institute of Technology POB 6980 76049 Karlsruhe Germany

Funding

European Research Council (Advanced Grant ‘COMREC’)

Cite This Article

Friedrich Fauser, Simon Schiml, Holger Puchta (2014). Both CRISPR/Cas‐based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. The Plant Journal, 79(2), 348-359. https://doi.org/10.1111/tpj.12554

Both CRISPR/Cas‐based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana

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