An Experimental Protocol for Teaching CRISPR/Cas9 in a Post-Graduate Plant Laboratory Course: An Analysis of Mutant-Edited Plants without Sequencing

Autor/inn/en	Mayta, Martín L.; Dotto, Marcela; Orellano, Elena G.; Krapp, Adriana R.
Titel	An Experimental Protocol for Teaching CRISPR/Cas9 in a Post-Graduate Plant Laboratory Course: An Analysis of Mutant-Edited Plants without Sequencing
Quelle	In: Biochemistry and Molecular Biology Education, 50 (2022) 5, S.537-546 (10 Seiten)Infoseite zur Zeitschrift PDF als Volltext Verfügbarkeit
Zusatzinformation	ORCID (Orellano, Elena G.)
Sprache	englisch
Dokumenttyp	gedruckt; online; Zeitschriftenaufsatz
ISSN	1470-8175
DOI	10.1002/bmb.21659
Schlagwörter	Graduate Study; College Science; Science Education; Science Instruction; Teaching Methods; Plants (Botany); Science Laboratories; Visualization; Laboratory Experiments + Suchen Sie Ihr Suchwort? Aufbaustudium; Graduiertenstudium; Hauptstudium; Naturwissenschaftliche Bildung; Teaching of science; Science education; Natural sciences Lessons; Naturwissenschaftlicher Unterricht; Teaching method; Lehrmethode; Unterrichtsmethode; Pflanze; Visualisation; Visualisierung; Laboratory work; Laborarbeit
Abstract	The CRISPR/Cas9 system is widely used for editing genes in various organisms and is a very useful tool due to its versatility, simplicity, and efficiency. To teach its principles to post-graduate students we designed a laboratory activity to obtain and analyze PDS3 mutants in "Arabidopsis thaliana" plants consisting of: 1) Design of guide RNAs using bioinformatics tools; 2) plant transformation (which is optional depending on the length of the course); 3) observation and evaluation of the mutant's phenotypes in the "Phytoene desaturase" (PDS3) gene, which exhibit an albino phenotype and different degrees of mosaicism in the editing events we evaluated; 4) PCR amplification of a fragment that includes the mutated region followed by analysis of single-stranded DNA conformation polymorphisms (SSCP) using native polyacrylamide gel electrophoresis and silver nitrate staining to detect changes in the amplicon sequence due to gene editing. Through SSCP, the students were able to distinguish between homozygous and heterozygous edited plants. A highlight feature of this protocol is the visualization and detection of the mutation/edition without sequencing the edited fragment. (As Provided).
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Erfasst von	ERIC (Education Resources Information Center), Washington, DC
Update	2024/1/01