The DOI matches its Crossref record; 11 of the 11 references it cites were found in a registry or an index.
Both CRISPR/Cas‐based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana
Abstract
Summary: Engineered nucleases can be used to induce site‐specific double‐strand breaks (DSB s) 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/C as9 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 (SSB s). 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 ADH 1 and TT 4 genes of A rabidopsis 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–Sce I. 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 C as9 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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Cites
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Cited by
Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., Wang, B., Yang, Z., Li, H., Lin, Y., et al. (2015) · Molecular Plant · doi:10.1016/j.molp.2015.04.007 · Cited by 2,369
A CRISPR/Cas9 toolkit for multiplex genome editing in plants
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Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion
Shimatani, Z., Kashojiya, S., Takayama, M., Terada, R., Arazoe, T., Ishii, H., Teramura, H., Yamamoto, T., Komatsu, H., Miura, K., et al. (2017) · Nature Biotechnology · doi:10.1038/nbt.3833 · Cited by 812
Svitashev, S., Young, J. K., Schwartz, C., Gao, H., Falco, S. C., & Cigan, A. M. (2015) · Plant Physiology · doi:10.1104/pp.15.00793 · Cited by 733
A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation
Lowder, L. G., Zhang, D., Baltes, N. J., Paul, J. W., Tang, X., Zheng, X., Voytas, D. F., Hsieh, T.-F., Zhang, Y., & Qi, Y. (2015) · Plant Physiology · doi:10.1104/pp.15.00636 · Cited by 707
Metadata
- Authors
- Fauser, F., Schiml, S., & Puchta, H.
- Venue
- The Plant Journal
- Publisher
- Wiley
- Published
- 2014-06-17
- Volume
- 79
- Issue
- 2
- Pages
- 348-359
- ISSN
- 0960-7412 (ISSN-L)
- 1365-313X
- OpenAlex
- W2017746652
- Cited by
- 713
- References
- 11
- FWCI
- 22.02
- Type
- journal article
- Language
- en
- Open access
- closed
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