ERIC Number: EJ1349444
Record Type: Journal
Publication Date: 2022
Pages: 10
Abstractor: As Provided
ISBN: N/A
ISSN: ISSN-1470-8175
EISSN: EISSN-1539-3429
Available Date: N/A
An Experimental Protocol for Teaching CRISPR/Cas9 in a Post-Graduate Plant Laboratory Course: An Analysis of Mutant-Edited Plants without Sequencing
Mayta, Martín L.; Dotto, Marcela; Orellano, Elena G.; Krapp, Adriana R.
Biochemistry and Molecular Biology Education, v50 n5 p537-546 Sep-Oct 2022
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.
Descriptors: Graduate Study, College Science, Science Education, Science Instruction, Teaching Methods, Plants (Botany), Science Laboratories, Visualization, Laboratory Experiments
Wiley. Available from: John Wiley & Sons, Inc. 111 River Street, Hoboken, NJ 07030. Tel: 800-835-6770; e-mail: cs-journals@wiley.com; Web site: https://bibliotheek.ehb.be:2191/en-us
Publication Type: Journal Articles; Reports - Descriptive
Education Level: Higher Education; Postsecondary Education
Audience: N/A
Language: English
Sponsor: N/A
Authoring Institution: N/A
Grant or Contract Numbers: N/A
Author Affiliations: N/A