{"title":"A simple, cost-effective, and efficient method for screening CRISPR/Cas9 mutants in plants","authors":"","doi":"10.1016/j.jplph.2024.154375","DOIUrl":null,"url":null,"abstract":"<div><div>The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing system is widely used for targeted mutagenesis in a growing number of plant species. To streamline the screening process for mutants, especially those generated from low-efficiency editing events, there is a need for a rapid, cost-effective, and efficient method. Although several screening methods have been developed to process initial samples, these methods often tend to be time-consuming, expensive, or inefficient when dealing with larger sample sizes. Here we describe a simple, rapid, low-cost, and sensitive screening method for screening CRISPR/Cas9 mutants called PCR-<em>Bsl</em> I-associated analysis (PCR-BAA). This method requires only standard PCR and <em>Bsl</em> I restriction enzyme digestion, as well as agarose gel electrophoresis analysis. This method is particularly well suited for the efficient screening of mutants from larger populations of transformants. The simplicity, low cost, and high sensitivity of the PCR-BAA method make it particularly suitable for rapid screening of CRISPR/Cas9-induced mutants, especially those from low-efficiency editing events.</div></div>","PeriodicalId":16808,"journal":{"name":"Journal of plant physiology","volume":null,"pages":null},"PeriodicalIF":4.0000,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of plant physiology","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0176161724002062","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"PLANT SCIENCES","Score":null,"Total":0}
引用次数: 0
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing system is widely used for targeted mutagenesis in a growing number of plant species. To streamline the screening process for mutants, especially those generated from low-efficiency editing events, there is a need for a rapid, cost-effective, and efficient method. Although several screening methods have been developed to process initial samples, these methods often tend to be time-consuming, expensive, or inefficient when dealing with larger sample sizes. Here we describe a simple, rapid, low-cost, and sensitive screening method for screening CRISPR/Cas9 mutants called PCR-Bsl I-associated analysis (PCR-BAA). This method requires only standard PCR and Bsl I restriction enzyme digestion, as well as agarose gel electrophoresis analysis. This method is particularly well suited for the efficient screening of mutants from larger populations of transformants. The simplicity, low cost, and high sensitivity of the PCR-BAA method make it particularly suitable for rapid screening of CRISPR/Cas9-induced mutants, especially those from low-efficiency editing events.
期刊介绍:
The Journal of Plant Physiology is a broad-spectrum journal that welcomes high-quality submissions in all major areas of plant physiology, including plant biochemistry, functional biotechnology, computational and synthetic plant biology, growth and development, photosynthesis and respiration, transport and translocation, plant-microbe interactions, biotic and abiotic stress. Studies are welcome at all levels of integration ranging from molecules and cells to organisms and their environments and are expected to use state-of-the-art methodologies. Pure gene expression studies are not within the focus of our journal. To be considered for publication, papers must significantly contribute to the mechanistic understanding of physiological processes, and not be merely descriptive, or confirmatory of previous results. We encourage the submission of papers that explore the physiology of non-model as well as accepted model species and those that bridge basic and applied research. For instance, studies on agricultural plants that show new physiological mechanisms to improve agricultural efficiency are welcome. Studies performed under uncontrolled situations (e.g. field conditions) not providing mechanistic insight will not be considered for publication.
The Journal of Plant Physiology publishes several types of articles: Original Research Articles, Reviews, Perspectives Articles, and Short Communications. Reviews and Perspectives will be solicited by the Editors; unsolicited reviews are also welcome but only from authors with a strong track record in the field of the review. Original research papers comprise the majority of published contributions.