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. 2021 May 31;22(2):e00106-21.
doi: 10.1128/jmbe.00106-21. eCollection 2021 Fall.

CRISPR-Cas9 Gene Editing in Yeast: A Molecular Biology and Bioinformatics Laboratory Module for Undergraduate and High School Students

Saumya M Sankaran  1 , Justin D Smith  2   3 , Kevin R Roy  2   3
Affiliations

CRISPR-Cas9 Gene Editing in Yeast: A Molecular Biology and Bioinformatics Laboratory Module for Undergraduate and High School Students

Saumya M Sankaran et al. J Microbiol Biol Educ. .

Abstract

CRISPR-Cas9 genome editing technology is widely used in scientific research and biotechnology. As this technology becomes a staple tool in life sciences research, it is increasingly important to incorporate it into biology curricula to train future scientists. To demonstrate the molecular underpinnings and some limitations of CRISPR-based gene editing, we designed a laboratory module to accompany a discussion-based course on genome editing for college and advanced high school biology students. The laboratory module uses CRISPR-Cas9 to target and inactivate the ADE2 gene in Saccharomyces cerevisiae so as to give red colonies, employing an inexpensive yeast model system with a phenotypic readout that is easily detectable without specialized equipment. Students begin by accessing the yeast ADE2 sequence in a genome database, applying their understanding of Cas9 activity to design guide RNA (gRNA) sequences, using a CRISPR analysis tool to compare predicted on- and off-target effects of various gRNAs, and presenting and explaining their choice of an optimal gRNA to disrupt the ADE2 gene. They then conduct yeast transformations using Cas9 and preselected gRNA plasmids with or without donor templates to explore the importance of DNA repair pathways in genome editing. Lastly, they analyze the observed editing rates across different gRNAs targeting ADE2, leading to a discussion of editing efficiency. This module engages students in experimental design, provides hands-on experience with CRISPR-Cas9 gene editing and collaborative data analysis, and stimulates discussion on the uses and limitations of CRISPR-based gene editing technology.

Keywords: CRISPR; CRISPR-Cas9; DNA repair; college biology lab; gene editing; high school biology lab; laboratory module; project-based lab; yeast.

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Figures

FIG 1
FIG 1
Flow chart of the experiment. The breakdown of laboratory activities by lab day reflects the schedule that we used. The schedule can be adapted as needed; notes next to arrows on the flow chart indicate required time intervals between laboratory components. The Optional Labs were designed but not taught due to course time constraints.
FIG 2
FIG 2
gRNA design platform. Shown is the process of designing CRISPR gRNAs to target the ADE2 gene using the Benchling web application (free for academic and educational use). (a) First, students design gRNAs against ADE2 by hand, applying their understanding of Streptococcus pyogenes Cas9 activity to identify protospacer-adjacent motif (PAM) sequences, gRNA sequences associated with the PAM, and the Cas9 cut site corresponding to the gRNA. (b) Using the Benchling CRISPR analysis tool, students first define Cas protein and target genome parameters to generate a list of possible gRNAs and then compare on- and off- target scores to choose optimized gRNAs for their experiment.
FIG 3
FIG 3
CRISPR-Cas9-mediated genome editing of the ADE2 gene in budding yeast. (a) The ADE2 gene is shown with the direction of transcription indicated by the black arrow. The four different gRNAs utilized in this module (g1, g2, g3, and g4) are depicted with their approximate locations relative to the start of the ORF. Cas9 cleavage of the ADE2 ORF with gRNA g1 can be repaired either through mutagenic NHEJ or through HDR. NHEJ ligates the broken ends with small indels. The indels block further cleavage by Cas9 and can also lead to inactivation of the ORF by a frameshift. HDR with cotransformed donor DNA leads to a precise deletion of the ORF. The molecular outcomes of the editing events can be interrogated by PCR using forward (F) and reverse (R) primers situated upstream and downstream of the ORF, respectively. See Appendix 1E in the supplemental material for more details. (b) Representative plate images of yeast transformations with each ADE2 gRNA with or without donor DNA. A no-gRNA control is used to demonstrate the colony yield obtained in the absence of genome editing. Note the clearly visible red pigment accumulating in colonies transformed with guides 1, 2, and 3, and how the addition of donor DNA impacts the size and color of the edited (red) colonies.
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