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.
Highlights
Increasing yield and quality of crops is the ultimate objective of plant breeding
CRISPR/Cas9 mainly utilizes single guide RNA to direct cleavage of the target DNA by the CAS9 protein, which generates double-stranded breaks (DSBs) at the target site that are usually repaired through nonhomologous end joining (NHEJ), an error-prone mechanism causing mutations in the target site [15,16,17]
Compared with zinc finger nucleases (ZFNs) and transcription activator-like effect nucleases (TALENs), the CRISPR/Cas9 gene editing system is simple in design and operation, and it has a higher efficiency of target site editing
Summary
Increasing yield and quality of crops is the ultimate objective of plant breeding. Conventional plant breeding has made a significant contribution to meet the increasing demands on crop products caused by rapid expansion of human population, but it will be more difficult to keep up with the increasing demands of humans and industries for the agricultural products while under the pressure of the global environmental challenges that we face. CRISPR/Cas mainly utilizes single guide RNA (sgRNA) to direct cleavage of the target DNA by the CAS9 protein, which generates double-stranded breaks (DSBs) at the target site that are usually repaired through nonhomologous end joining (NHEJ), an error-prone mechanism causing mutations in the target site [15,16,17] This technology has been applied in Arabidopsis [18,19], rice [20,21], wheat [12], corn [22,23], tobacco [10,24], tomato [25], Sorghum bicolor [26], and other monocotyledonous and dicotyledonous plants [27,28,29,30] to generate mutants for investigation of gene function and new germplasms for crop breeding. Our major aim is to investigate the feasibility of a simple CRISPR/Cas gene editing system in targeted mutagenesis in cotton, and in future, to have transgenic cottons with different types of mutations in a gene with a potential role in fiber development for functional characterization
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