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Highly Efficient Targeted Gene Editing in Upland Cotton Using the CRISPR/Cas9 System

1
The Key Laboratory of Oasis Eco-Agriculture, College of Agriculture, Shihezi University, Shihezi 832000, Xinjiang, China
2
Key Laboratory of Plant Secondary Metabolism and Regulation of Zhejiang Province, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310016, Zhejiang, China
3
CSIRO Agriculture and Food, GPO Box 1700, Canberra 2601, Australia
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(10), 3000; https://doi.org/10.3390/ijms19103000
Received: 27 June 2018 / Revised: 28 September 2018 / Accepted: 28 September 2018 / Published: 1 October 2018
(This article belongs to the Special Issue Genome Editing in Plants)
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Abstract

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to be able to induce highly efficient mutagenesis in the targeted DNA of many plants, including cotton, and has become an important tool for investigation of gene function and crop improvement. Here, we developed a simple and easy to operate CRISPR/Cas9 system and demonstrated its high editing efficiency in cotton by targeting-ALARP, a gene encoding alanine-rich protein that is preferentially expressed in cotton fibers. Based on sequence analysis of the target site in the 10 transgenic cottons containing CRISPR/Cas9, we found that the mutation frequencies of GhALARP-A and GhALARP-D target sites were 71.4–100% and 92.9–100%, respectively. The most common editing event was deletion, but deletion together with large insertion was also observed. Mosaic mutation editing events were detected in most transgenic plants. No off-target mutation event was detected in any the 15 predicted sites analyzed. This study provided mutants for further study of the function of GhALARP in cotton fiber development. Our results further demonstrated the feasibility of use of CRISPR/Cas9 as a targeted mutagenesis tool in cotton, and provided an efficient tool for targeted mutagenesis and functional genomics in cotton. View Full-Text
Keywords: CRISPR/Cas9; genome editing; cotton; GhALARP; mutation CRISPR/Cas9; genome editing; cotton; GhALARP; mutation
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Zhu, S.; Yu, X.; Li, Y.; Sun, Y.; Zhu, Q.; Sun, J. Highly Efficient Targeted Gene Editing in Upland Cotton Using the CRISPR/Cas9 System. Int. J. Mol. Sci. 2018, 19, 3000.

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