Online Archive of University of Virginia Scholarship
An effective and inexpensive strategy to identify multiplex CRISPR-edited plants368 views
Author
Li, Renyu, Biology - Graduate School of Arts and Sciences, University of Virginia
Advisors
Danna, Cristian, AS-Biology, University of Virginia
Abstract
Since its first introduction in 2013, CRISPR-Cas9 has become the preferred gene targeting tool to produce loss-of-function mutants in plants. In spite of the high specificity and ease of use, the identification of CRISPR-edited plants has remained a time consuming and onerous process. I have developed and tested an easy-to-use and inexpensive strategy to select for multiplex CRISPR mutagenized Arabidopsis plants. This strategy is based on targeting the gene/s of interest simultaneously with a proxy for CRISPR-Cas9 activity: an endogenous gene that produces an easy-to-detect visible phenotype. To test this strategy, I have chosen Arabidopsis gene JAR1, GL1, EIN2 as the candidate proxies. I have tested the T2 progeny of independent T1 plants harboring CRISPR/Cas9 and successfully identified plants where the visible marker and the genes of interest were simultaneously edited at a high frequency. The co-editing frequency ranged from 55.6% to 93.75% for two genes, and 14.3% to 50% for three genes, depending on the T1 progeny tested and the proxy gene of choice. The visual phenotype selection provides a narrow pool of plants to analyze, hence increasing the recovery frequency while decreasing the cost of identifying mutants. This selection strategy also offers a framework to similarly facilitate the identification of CRISPR-edited plants in other plant species with more complex polyploid genomes where multiplex mutants are essential for studying gene function.
Li, Renyu. An effective and inexpensive strategy to identify multiplex CRISPR-edited plants. University of Virginia, Biology - Graduate School of Arts and Sciences, MS (Master of Science), 2019-04-24, https://doi.org/10.18130/v3-r8zy-1v33.