CRISPR Gene Editing in Yeast: An Experimental Protocol for an Upper‐Division Undergraduate Laboratory Course

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) are a revolutionary tool based on a bacterial acquired immune response system. CRISPR has gained widespread use for gene editing in a variety of organisms and is an increasingly valuable tool for basic genetic research, with far-reaching implications for medicine, agriculture, and industry. This lab is based on the premise that upper division undergraduate students enrolled in a Life Sciences curriculum must become familiar with cutting edge advances in biotechnology that have significant impact on society. Toward this goal, we developed a new hands-on laboratory exercise incorporating the use of CRISPR-Cas9 and homology directed repair (HDR) to edit two well-characterized genes in the budding yeast, Saccharomyces cerevisiae. The two genes edited in this exercise, Adenine2 (ADE2) and Sterile12 (STE12) affect metabolic and developmental processes, respectively. Editing the premature stop codons in these genes results in clearly identifiable phenotypes that can be assessed by students in a standard laboratory course setting. Making use of this basic eukaryotic model organism facilitates a laboratory exercise that is inexpensive, simple to organize, set up, and present to students. This exercise enables undergraduate students to initiate and follow-up on all stages of the CRISPR gene editing process, from identification of guide RNAs, amplification of an appropriate HDR fragment, and analysis of mutant phenotypes. The organization of this protocol also allows for easy modification, providing additional options for editing any expressed genes within the yeast genome to produce new mutations, or recovery of existing mutants to wild type.

An undergraduate laboratory module that uses the CRISPR/Cas9 system to generate frameshift mutations in yeast

“…Because the yeast genome can be modified in various ways with relative ease, instructors can choose a set of experiments that best aligns with their teaching objectives for a particular course. While this lab module can show students how the CRISPR/Cas9 system can generate random frameshift mutations with NHEJ, a different lab module can teach students how the CRISPR/Cas9 system can generate specific mutations with HDR. Moving forward, it will be important to create a course‐based undergraduate research experience that will allow students to develop a hypothesis and to test it by designing their own gRNAs and carrying out CRISPR.…”

“…Using yeast as a model organism to teach CRISPR‐associated concepts to undergraduate students is ideal because it is inexpensive, easy to use, and provides an opportunity to perform gene editing in a eukaryotic system. Recently, Sehgal and colleagues used HDR and CRISPR/Cas9 in yeast to generate premature stop codons that were identified by screening colony phenotypes . Here, we describe a new undergraduate lab module that utilizes NHEJ and CRISPR/Cas9 in yeast to induce frameshift mutations that are identified by selection.…”

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.…”

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

“…[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.…”