An undergraduate laboratory experience using CRISPR‐cas9 technology to deactivate green fluorescent protein expression in Escherichia coli

Abstract

Undergraduates learn that gene editing in diverse organisms is now possible. How targeted manipulation of genes and genomes is utilized in basic science and biomedicine to address biological questions is challenging for undergraduates to conceptualize. Thus, we developed a lab experience that would allow students to be actively engaged in the full process of design, implementation of a gene editing strategy, and interpretation of results within an 8-week lab period of a Genetics course. The laboratory experience combines two transformative biotechnology tools; the utilization of green fluorescent protein (GFP) as a diagnostic marker of gene expression and the fundamentals and specificity of Clustered Regularly Interspaced Short Palindromic Repeats-cas9 (CRISPR-cas9) gene editing in bacterial cells. The students designed and constructed plasmids that express single guide RNA targeted to GFP, expressed the sgRNA and cas9 in bacteria cells, and successfully deactivated GFP gene expression in the bacterial cells with their designed CRISPR-cas9 tools. Student assessment revealed most students achieved student learning objectives. We conclude this lab experience is an effective and accessible method for engaging students in the scientific practices, knowledge and challenges revolving targeted CRISPR-cas9 gene manipulation. © 2019 International Union of Biochemistry and Molecular Biology, 47(2): 145-155, 2019.

“Designer babies?!” A CRISPR‐based learning module for undergraduates built around the CCR5 gene

“…To model formation of gRNA‐cas9‐target DNA complexes, students were tasked to hand draw how all these components interact, labeling important components and sites including their PAM sequence, the location of the cas9 cut site, the annealing of the gRNA to its specific target nucleotide sequence. We intentionally had students draw this out based on our prior teaching experiences where assessment revealed students found it challenging to conceptualize the action and polarity of gRNA and cas9 complex with two complementary DNA strands (target and non‐target strands) of the target gene 19 …”

“…We intentionally had students draw this out based on our prior teaching experiences where assessment revealed students found it challenging to conceptualize the action and polarity of gRNA and cas9 complex with two complementary DNA strands (target and non-target strands) of the target gene. 19 Students were prompted to discuss why this specific PAM associated with the target sequence was chosen of all possible PAM sequences of (5 0 NGG3 0 ) as a quick "Find" of "GG" highlights in yellow all the GGs present in the sequence (yellow boxes, Figure 5a), and students observe that there are many potential PAM sequences present throughout the gene. The discussion led the class to conclude that this specific 5 0 NGG3 0 was chosen of many potential 5 0 NGG3 0 because of the necessity for cas9 cut the CCR5 genomic DNA in close proximity to the physical location of the desired nucleotide change that could potentially give rise to Δ32 allele variant.…”

“…[10][11][12][13][14] Traditionally, instruction has relied upon these core molecular concepts being reinforced in a laboratory setting, where these misconceptions may be addressed through hands-on application-based lab work. CRISPR-cas-based methodologies have been used successfully to train students in fundamental molecular biology skills 15 and in a variety of model organisms, including yeast, [16][17][18] Escherichia coli, 19 Drosophila melanogaster, 20 and Arabidopsis thaliana. 21 However, not all undergraduate teaching laboratories are equipped to sufficiently perform gene editing experiments in live organisms and conduct the subsequent molecular and phenotypic analysis, let alone provide high impact course-based undergraduate research experiences (CUREs) for students using the technologies.…”

Bringing immersive science to undergraduate laboratory courses using CRISPR gene knockouts in frogs and butterflies

“…While most biology and pre-medical students leave college with a lecture-based understanding of CRISPR-related techniques, few will have had the opportunity to perform it in a laboratory. There are increasing numbers of classroom laboratory modules involving CRISPR experiments performed in bacteria (Pieczynski et al, 2019), yeast (Sehgal et al, 2018) and Drosophila (Adame et al, 2016), signaling increasing incorporation of this important technology into science education (Wolyniak et al, 2019). To enrich this repertoire of pedagogical approaches, we devised undergraduate laboratory courses involving gene editing in live animals.…”

A Scalable CURE Using a CRISPR/Cas9 Fluorescent Protein Knock-In Strategy in Caenorhabditis elegans

“…Genome editing with CRISPR/Cas9 technology has advanced from the lab bench to clinical application, with the first phase 1 and 2 trials underway to treat β-thalassemia (NCT03655678) and sickle cell disease (NCT03745287). Undergraduate laboratory course introductions to CRISPR/Cas9 commonly feature bacteria or yeast (1, 2) but have also been successfully implemented as course-based undergraduate research experiences (CUREs) featuring zebrafish, at the University of Alabama at Birmingham (3), and Caenorhabditis elegans , at Pomona College (using the SapTrap method [4], different than the two-plasmid system presented here [Sara K. Olson, personal communication]).…”