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An undergraduate laboratory module that uses the CRISPR/Cas9 system to

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Article
An Undergraduate Laboratory Module That
Uses the CRISPR/Cas9 System to Generate
Frameshift Mutations in Yeast S
From the †Biology Department, Suffolk University, Boston, Massachusetts,
‡Department of Biochemistry and Molecular Biology, University of
Massachusetts Amherst, Amherst, Massachusetts
Abstract
The CRISPR/Cas9 system is a powerful tool for gene editing
and it has become increasingly important for biology students
to understand this emerging technique. Most CRISPR laboratory
teaching modules use complex metazoan systems or mamma-
lian cell culture which can be expensive. Here, we present a
lab module that engages students in learning the fundamentals
of CRISPR/Cas9 methodology using the simple and inexpensive
model system, Saccharomyces cerevisiae. Students use
CRISPR/Cas9 and nonhomologous end joining to generate
frameshift insertion and deletion mutations in the CAN1 gene,
which are easily selected for using media plates that have
Keywords: Active learning; biotechnology education; DNA repair;
laboratory exercises; molecular biology; mutagenesis; new course
development
Introduction
The ability to make genomic changes has been a tantalizing
prospect for many years in the scientific community with
many potential applications in biomedical research and thera-
peutic intervention. In the early 1980s, it was discovered that
homologous recombination could be used to make precise
changes to specific genes in cultured mammalian cells [1]. A
decade later, nucleases were designed to make targeted cuts in
the genome, which could introduce modifications by activating
endogenous DNA repair mechanisms [2, 3]. Although these
advancements allowed scientists to manipulate the genomes of
different species, technical and logistical limitations precluded
Volume 00, Number 00, Month 2019, Pages 1–8
*To whom correspondence should be addressed. Tel.: 617-573-8600.
E-mail: edewaal@suffolk.edu and Tel.: 617-573-8249; Fax: 617-573-
8245. E-mail: cnpeterson@suffolk.edu
S Additional Supporting Information may be found in the online version
of this article.
Received 20 February 2019; Revised 22 May 2019; Accepted 2
June 2019
DOI 10.1002/bmb.21280
Published online 00 Month 2019 in Wiley Online Library
(wileyonlinelibrary.com)
Biochemistry and Molecular Biology Education
Eric de Waal†*
Thomas Tran†‡
Domenic Abbondanza†
Arup Dey†
Celeste Peterson †*
canavanine. DNA sequencing is also performed to determine
what type of mutation occurred in gene-edited cells. This easy to
implement set of experiments has been run as both a 5-week
and a shorter 3-week lab module. Learning assessments dem-
onstrate increased understanding in CRISPR-related concepts
as well as increased confidence using molecular techniques.
Thus, this CRISPR/Cas9 lab module can be added to an existing
Genetics, Microbiology, or Molecular Biology lab course to help
undergraduate students learn current gene editing techniques
with limited effort and cost. © 2019 International Union of Bio-
chemistry and Molecular Biology, 00(00):1–8, 2019.
their widespread use in the lab and clinic [4]. However,
the discovery of Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR) and CRISPR-associated (Cas)
genes has revolutionized the field of gene editing because
CRISPR tools are cheap, easy to use, and efficient at making
targeted alterations to genomes in living cells [5].
The CRISPR sequences and Cas proteins are components
of an adaptive system that targets foreign DNA in many bacte-
rial species [6, 7]. CRISPR sequences, acquired from past
infections, represent the memory of the system. The Cas pro-
teins process CRISPR sequences into small RNAs which target
infectious DNA molecules through complementary base-
pairing. In the bacterium Streptococcus pyogenes, an endo-
nuclease (Cas9) interacts with a CRISPR RNA (crRNA) and a
trans-activating crRNA (tracrRNA) to target foreign DNA
molecules [8]. After crRNA binding to the foreign DNA with
complementary base pairing, the Cas9 generates a double-
strand break (DSB) leading to its degradation.
To convert the complex prokaryotic immune system into
a straightforward gene editing tool, biomedical engineers
fused the crRNA and tracrRNA to form a synthetic guide
RNA (gRNA). This simple gRNA can interact with Cas9 and
recognize a 20 base-pair (bp) sequence in chromosomal
DNA [8]. Once the Cas9/gRNA complex binds to a genomic
1

sequence, a DSB is generated which activates endogenous
DNA repair mechanisms that will facilitate the targeted
genetic modification. If donor DNA is present, then homology
directed repair (HDR) is used to make a specific genetic
change where the DSB occurred. On the other hand, if there is
no donor DNA present when Cas9 cuts the DNA then non-
homologous end joining (NHEJ) will generate either deletions
or insertions at the site of the DSB. As the CRISPR/Cas9 system
can be used to target virtually any genome location in any spe-
cies with relative ease, it has become a powerful tool that has
allowed scientists to delve into new scientific questions [9],
generate genetically modified organisms without inserting for-
eign transgenes [10], and develop novel therapeutic treat-
ments for previously incurable genetic diseases [11].
A central responsibility of educators at universities and col-
leges is to incorporate the latest technological advances into
teaching labs, and thus it is important that undergraduate stu-
dents have first-hand experience with the CRISPR/Cas9 system.
While some teaching labs using CRISPR/Cas9 gene editing have
already been designed [12–14], they typically require complex
and expensive cell culture systems that may not be feasible for
smaller, teaching-centered institutions of higher learning. 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 [15]. 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. In addition, students can visualize
the different deletion and insertion mutations generated by
CRISPR/Cas9 using PCR and DNA sequencing. The various exper-
iments in this lab module are focused on four learning objectives:
1. Describe how the CRISPR/Cas system works in prokary-
otic cells
2. Explain how CRISPR/Cas9 can be used to generate
targeted genetic modifications
3. Increase students’ confidence in using molecular biology
techniques for scientific research
4. Increase confidence in understanding the CRISPR/Cas9 system
Notably, this lab module is based on an existing protocol
developed by George Church and colleagues [16], so all nec-
essary reagents are common-source items that can be easily
purchased. Therefore, any institution that has basic labora-
tory supplies necessary for culturing yeast cells can incorpo-
rate these experiments into their curriculum.
Lab Module Organization and
Assessment
This module has been taught over four semesters in a Genetics
lab (BIO L274) that supplements the Genetics lecture (BIO 274)
2 Using CRISPR and NHEJ to Teach Students About Gene Editing
Biochemistry and
Molecular Biology Education
at Suffolk University (see outline in Supporting Information File
1). Each weekly, 3-hour lab has between 4 and 20 undergradu-
ate students, ranging from sophomores to seniors, who work
in pairs for all experiments. The full-length CRISPR/Cas9 lab
module is composed of five labs, but a shorter 3-week version
can be used to focus on concepts underlying the CRISPR/Cas9
system (outlined in Fig. 1). This shorter version has also been
successfully run at Suffolk University.
The experimental objective is to induce a frameshift
mutation at the endogenous CAN1 gene that codes for an
arginine transporter in yeast. The module starts by activating
Cas9 expression, which is controlled by a galactose-inducible
promoter, in two different yeast strains: 1) yeast that have a
plasmid containing the Cas9 gene (Cas9 cells) and 2) yeast
that have a plasmid containing the Cas9 gene and a plasmid
with a gRNA for the CAN1 gene (Cas9 + CAN1 gRNA cells).
Yeast containing only the Cas9 plasmid cannot alter the
CAN1 gene and serves as a negative control whereas yeast
that have both the Cas9 and CAN1 gRNA plasmids are able
to undergo gene editing at the CAN1 gene.
Selection for yeast with frameshift mutations at the CAN1
gene can be done using culture media containing canavanine,
a toxic arginine analog. In wild type cells, canavanine is impo-
rted through the CAN1 transporter and causes cell death. Cells
with functional transporters will die in the presence of
canavanine while those with an edited CAN1 gene will grow
and form colonies. To determine if gene editing occurred,
genomic DNA from canavanine-resistant colonies is used for
PCR amplification of the CAN1 gene and sequence analysis.
The lab module ends with a sequence alignment between
a wild-type CAN1 gene sequence and a CRISPR-edited
CAN1 sequence so students can visualize the different
types of insertion or deletion mutations that enabled
canavanine resistance. The shorter version of the module
(Fig. 1) does not include the genomic DNA isolation or
PCR, so students are given previously generated CAN1
sequences (available upon request) from canavanine-
resistant colonies to analyze.
Several steps are taken to help students learn about
the CRISPR/Cas9 approach and meet the module’s learn-
ing objectives. First, students are instructed to write out
the protocol for each experiment before coming to the lab
and ask questions about the protocol before beginning
the experiment. Students also take prelab quizzes at the
beginning of each class to assess their knowledge on tech-
niques and concepts. Finally, at the end of the lab mod-
ule, each student must answer a set of questions to
demonstrate an in-depth understanding for the rationale
and overall objective of the CRISPR/Cas9 lab module.
Materials and Methods
Protocols and preparation notes for all labs and experi-
ments can be found in Supporting Information Files 2 and 3.
 Schematic of different labs in CRISPR/Cas9 lab module. In five different labs, Cas9 and CAN1 gRNA plasmids are used to
FIG 1 generate frameshift mutations at the endogenous CAN1 gene in haploid yeast. A 3-week version that focuses on the
mechanisms of gene editing is also outlined. [Color figure can be viewed at wileyonlinelibrary.com]

Yeast Strain and Media
The Saccharomyces cerevisiae strain that was used for the gene
editing lab was BY4741 (ATCC). The parental BY4741 strain was
grown in yeast extract peptone dextrose (YPD) media before
transformation. After the transformation, the strains were prop-
agated in the appropriate synthetic complete media (SC media,
Sunrise Science) that was lacking the auxotrophic compound
which is complemented by the Cas9 (leucine) and gRNA (uracil)
plasmids. Canavanine agar plates contained SC-arginine-leucine
media, 40 μg/ml L-canavanine (Sigma), and agar.
Cas9 and CAN1 gRNA Plasmids
The two plasmids were developed in a previous publication
[16], and can be purchased from Addgene. The Cas9 plasmid
(Plasmid #43804) has a galactose-inducible promoter (Gal-L)
that controls the expression of the Cas9 gene, and a leucine
selectable marker, LEU2. The CAN1 gRNA plasmid (Plasmid
#43803) contains the gRNA for CAN1 and an uracil selectable
marker, URA3.
shaker at 30
C overnight in YPD media. The following morn-
ing, 2.5 ml of culture was transferred to 30 ml of fresh YPD
media and the cells were propagated for 4–5 hours at 30
C.
Cells were collected by putting the 15 ml culture in two 50 ml
conical tubes and spinning at 3,000 × g for 5 minutes. The cell
pellets were washed once with 10 ml water then resuspended
in 2 ml of 1 M sorbitol. After spinning again at 3,000 × g, the
cells were resuspended in 1 ml of 500 mM Lithium Ace-
tate/10 mM Dithiothreitol (Sigma), incubated at room tempera-
ture for 5 minutes, and centrifuged at 3,000 × g for 5 minutes.
The cell pellet was washed once in 1 ml of 1 M sorbitol then
resuspended in 500 μl of 1 M sorbitol. Plasmid DNA (~200 ng
per transformation) was added to a 50 μl aliquot of competent
cells. Cells were electroporated at 1.5 kV, 25 μF, 200 mA. After
electroporation, 1 ml of YPD was added to the cuvette, cells
were transferred to a microcentrifuge tube, incubated in a
roller drum for 1 hour at 30
C, and plated on selective media
(SC-leucine for Cas9 cells or SC-leucine and uracil for Cas9 +
CAN1 gRNA cells).

Transformation of Plasmids into Yeast Activating Cas9 Expression with Galactose

The Cas9 and CAN1 gRNA plasmids were transformed into the Cas9 and Cas9 + CAN1 gRNA cells were grown in 5 ml SC-
BY4741 yeast strain using a lithium acetate transformation leucine media containing 2% glucose overnight and washed
protocol [17]. Briefly, a 15 ml culture of yeast was grown in twice with 10 ml water. After the second wash, cells were

de Waal et al. 3

resuspended in 10 ml SC-leucine media containing 2% galac-
tose/1% raffinose (Sigma). Cells were diluted to OD660 = 0.3
using SC-leucine media with 2% galactose/1% raffinose and
incubated for at least 24 hours at 30
C. Cells can be stored
at 4
C for at least 1 week before plating.
Selecting for Genome-edited Cells with Canavanine
Plates
SC-leucine and arginine plates containing L-canavanine were
used to select for yeast cells that have an inactivating mutation
at the CAN1 gene. After Cas9 and Cas9 + CAN1 gRNA cells
were incubated in media containing galactose for at least
24 hours, they were diluted to OD660 = 0.3 using SC-leucine
media with 2% glucose. Selection was performed by plating
100 μl of each diluted strain (~4-fold dilution) on SC-leucine
and arginine plates that contain 40 μg/ml L-canavanine. Cells
were grown on canavanine plates at 30
C for 2 days.
Amplification of the CAN1 Gene
Genomic DNA from canavanine-resistant Cas9 + gRNA cells
was extracted using the Yeast DNA isolation kit (Thermo Fisher)
and PCR was used to amplify the CAN1 gene. The following was
used in each 40 μl PCR reaction: 14 μl sterile water, 20 μl
Taq 2X Master Mix (New England Biolabs), 2 μl forward primer
(50 -GTGGTTTCCGGGTGAGTCAT-30 ), 2 μl reverse primer
(50 -GAAGCGACCCAGAACTCGAA-30 ), and ~200 ng genomic
DNA. The PCR ran for 35 cycles using the following three steps:
94
C for 30 seconds, 60
C for 30 seconds, and 68
C for
1 minute. At the end of the 35 cycles there was a final extension
of 68
C for 5 minutes. Once the PCR reaction was completed,
20 μl was loaded into a 1% agarose gel to check for the presence
of a 1,062 bp PCR product.
Sequencing the CAN1 PCR Product
The remaining 20 μl from the PCR sample was purified follow-
ing the manufacturer’s instructions with the E.Z.N.A. Cycle
Canavanine plates with Cas9 or Cas9 + CAN1 gRNA cells. Selective media plates containing canavanine are used to
FIG 2 select for cells that have an inactivating mutation at the CAN1 gene. [Color figure can be viewed at
wileyonlinelibrary.com]
4 Using CRISPR and NHEJ to Teach Students About Gene Editing
Biochemistry and
Molecular Biology Education
Pure Kit (Omega). After purification, 200–500 ng was diluted
in 10 μl water. About 2 μl of 1 μM sequencing primer (50 -
TTCAAAAGAAGACGCCGACA-30 ) was added to each sample,
and samples were sent in for sequencing (GeneWiz). The chro-
matogram file was viewed using free computer software
(FinchTV). Frameshift mutations at the CAN1 gene were iden-
tified by aligning the PCR product sequence to the wild-type
CAN1 sequence using Clustal Omega (www.ebi.ac.uk/Tools/
msa/clustalo/).
Assessment of Learning Objectives
To determine how the module affected student learning and atti-
tudes toward research, pretesting and post-testing on the learn-
ing objectives was performed. Multiple-choice questions were
used to assess whether students understood the mechanisms of
the CRISPR/Cas system in prokaryotes, and the concepts under-
lying CRISPR/Cas9 gene editing. In addition, attitudinal surveys
were filled out by students to assess their confidence in using
molecular biology techniques for scientific research with an
emphasis on using CRISPR/Cas9 for genome modification. All
testing was anonymous, voluntary, and ungraded (assessment
strategies were evaluated and approved by the Institutional
Review Board at Suffolk University).
Results
Selecting for Cells That Have Undergone Gene
Editing at CAN1
Targeting the CAN1 gene works particularly well as mutated
CAN1 cells can be selected for on canavanine plates, even if
gene editing occurs at a low frequency [18]. In Labs 1 and
2, students activated expression of Cas9 in yeast strains with
and without the gRNA for 24 hours and subsequently plated
them on canavanine plates (Fig. 1). In Lab 3, students were
given their two canavanine plates from the previous lab to

Agarose with successful CAN1 PCR amplification.
FIG 3 Genomic DNA from canavanine-resistant Cas9 +
CAN1 gRNA colonies was used to amplify a 1,062 bp
product within the coding region of CAN1. Three PCR
samples were run on a 1% agarose gel with a DNA
ladder.
determine if genome editing had occurred in Cas9 + CAN1
gRNA cells. There was some variation among the student
groups due to pipetting and plating techniques, but most stu-
dents observed ~1,000-fold difference in the number of colo-
nies between the Cas9 and Cas9 + CAN1 gRNA cells (Fig. 2).
After the students discussed the results with their lab partner,
they were asked to explain the results for both plates in the
conclusion section of their lab notebook.
There are a few issues to consider during the first three
labs. The efficiency of gene editing is largely dependent on
mitotic proliferation during growth with galactose. As long
as the yeast have been recently transformed with plasmids
or streaked out from freezer stocks, then cells should have
robust growth in galactose media which will enable efficient
gene editing. We typically have >90% of the student groups
observe results similar to Fig. 2. The most common problem
associated with Labs 1–3 is cross-contamination between
Cas9 and Cas9 + CAN1 gRNA cultures that is often caused
by not changing pipette tips in Labs 1 and/or 2. This mistake
results in a large number of colonies on the canavanine
plate with Cas9 cells, so students should constantly be
reminded about the importance of changing pipette tips
every time they use the micropipette.
Amplification of Endogenous CAN1 in Canavanine-
resistant Cells Using PCR
To confirm that the increased number of colonies on the
canavanine plate with Cas9 + CAN1 gRNA cells was due to
CRISPR/Cas9 gene editing at the CAN1 gene and not a sponta-
neous point mutation, students performed a sequence analysis
of the amplified CAN1 gene (Fig. 1). The primers were
designed to amplify a 1,062 bp product that contains the 20 bp
sequence targeted by the CAN1 gRNA. After PCR, students
used half the sample (20 μl) for agarose gel electrophoresis,

Sequence alignment showing frameshift mutations at CAN1 are generated by the Cas9/CAN1 gRNA complex. (A) Wild-
FIG 4 type CAN1 (WT CAN1) sequence aligned with student-generated PCR sequences. The 20 bp sequence within the CAN1
gRNA that is used to localize Cas9 at the CAN1 locus is indicated by a black box. A “GG” insertion mutation is directly
below the wild-type sequence and a deletion mutation is indicated by dashes. (B) Table of different frameshift mutations
at CAN1 that have been generated by students.

de Waal et al. 5
 Biochemistry and
Molecular Biology Education

while the other half was column-purified for sequencing. We
typically observe a high success rate for this PCR, as demon-
strated by a band in the agarose gel around 1,000 bp (Fig. 3).
After the class examined the agarose gel and discussed the
results, each group of students selected one sample to be sent
in for sequencing. Because all groups of students performed
two PCRs (one for each student), each group had a backup
sample in case one of their reactions was not successful.
Although successful PCR amplification of CAN1 is com-
mon in Lab 4, there are certain precautions to take for this
lab. We have observed a decrease in the success rate of PCR
if the DNA isolation kit is old (>1 year), not stored at 4
C for
long periods of time, or if the students did not completely
resuspend their pellets in the lysis buffer. Another common
pitfall associated with this lab is poor loading of the sample
into the agarose gel, which can make it difficult to deter-
mine if amplification occurred. We typically have students
practice loading dyed water samples in an alternate gel
before loading their PCR sample into an agarose gel that
contains a molecular weight marker. This practice improves
the resolution of bands and gives students an opportunity to
increase their proficiency in pipetting and gel loading.
Students are also prone to make pipetting mistakes when
setting up PCRs, but the primers can amplify CAN1 even
with minor pipetting errors.
Sequence Alignment with PCR Product and
Wild-type CAN1
In the final lab of this module, the sequence of the amplified
CAN1 gene was analyzed to determine if it was altered by
the CRISPR/Cas9 gene editing system. Students were given
the raw chromatogram file of the sequenced PCR product to
visualize the peaks and assess the overall quality of the DNA
sequence using FinchTV software. Then, the students down-
loaded the wild-type CAN1 sequence from yeastgenome.org
and performed a sequence alignment using Clustal Omega
with their PCR sequence. In our experience, over 90% of
high-quality sequences generated by students have a frame-
shift mutation within or near the 20 bp sequence that is
targeted by the CAN1 gRNA (Fig. 4A). The frameshift muta-
tions induced by the CAN1 gRNA were mostly deletions of
various sizes (1–23 bp), but small insertions (1–2 bp) were
also observed (Fig. 4B).

(a) 1.0
Pre
Post
Ratio Correct

0.5

0.0
Objective 1: Objective 2:
CRISPR in prokaryotes Gene editing with CRISPR

(b) 5.0

4.0

3.0
Mean Score

2.0

1.0

0.0
Objective 3: Objective 4:
Confidence in conducting research Confidence in understanding CRISPR

Assessment of learning objectives for CRISPR lab module. (A) Multiple-choice questions were used to evaluate compre-
FIG 5 hension of concepts associated with CRISPR before (Pre) and after (Post) participating in the lab module. (B) Attitudinal
surveys were used to evaluate confidence before and after the lab module.

6 Using CRISPR and NHEJ to Teach Students About Gene Editing

 As long as students had PCR sequences with good peaks
and cadence, there were no significant problems with Lab
6. Poor quality DNA sequences are typically caused by an
error in purifying the PCR product, so measuring the quality
and concentration of purified DNA with a spectrophotome-
ter can identify samples that may be problematic. We often
saw the same deletion mutation between groups, but, as a
class, there is always a variety of insertion and deletion
mutations (Fig. 4). It is helpful to write out the list of muta-
tions during the lab, including the type (insertion or dele-
tion) and the number of base pairs involved. Students can
be asked to explain why none of the deletion/insertion base
pair numbers include multiples of three and thereby learn
about reading frame and in-frame deletions.
Evaluation of Learning Objectives
A total of 58 students from four different sections of the
genetics lab participated in assessment testing. An assess-
ment with multiple-choice questions (Supporting Informa-
tion File 4) was given to students at the beginning and end
of the lab module to evaluate whether they developed an
increased understanding of how the CRISPR/Cas9 system
works in prokaryotic cells as well as how CRISPR/Cas9 can
be used for gene editing. A large improvement in understand-
ing CRISPR in prokaryotes was evidenced by an increase
(~63%) in correct answers on multiple-choice questions cover-
ing this topic at the end of the lab module (Fig. 5A). Students
also answered multiple-choice questions regarding the use of
the CRISPR/Cas9 system in gene editing. Although there was
an increase in correct answers on these multiple-choice ques-
tions after the lab module, the increase was modest (~15%).
The low amount of improvement was most likely due to the
fact that this topic was previously covered in the lecture por-
tion of the genetics course, which led to a higher percentage of
correct answers at the beginning of the lab module (Fig. 5A).
Collectively, these results indicate that these experiments
increase student confidence in CRISPR/Cas9 methodology,
which translates into an improved understanding of how the
CRISPR system works in prokaryotic cells as well as how it can
be used for editing the genome.
To assess whether this lab module increased confidence
in scientific research and CRISPR/Cas9 methodology students
were given a paper survey (Supporting Information File 5) at
the beginning and end of the lab module that had a series of
statements. Students were asked to rate each statement
using a scale of 1–5 with five indicating high confidence and
one indicating no confidence. Overall, students indicated a
high level of confidence in conducting scientific research at
the beginning of the lab module (Fig. 5B). Although confi-
dence in conducting research was increased at the end of the
lab module (Fig. 5B) the improvement was modest (~7%) due
to the high scores in the initial assessment. The same paper
survey also had statements designed to evaluate student atti-
tudes toward understanding the CRISPR/Cas9 system. In this
instance, a large increase in confidence (~37%) was observed
de Waal et al.
at the end of the lab module indicating a substantial improve-
ment in student attitudes toward the basic concepts associ-
ated with CRISPR/Cas9 gene editing (Fig. 5B).
Conclusions and Future Directions
Due to the ever-growing importance of gene editing, it is critical
that undergraduate students develop an understanding of the
principles and mechanisms underlying this burgeoning technol-
ogy. The lab module described here has proven to be effective
in explaining how the CRISPR/Cas9 uses NHEJ to generate
targeted frameshift mutations. It also provides hands-on expe-
rience with a variety of lab techniques that are used to rein-
force concepts associated with the CRISPR/Cas9 system and
increase student confidence in conducting scientific research
outside of a classroom setting. The gene editing experiments
used in this lab module have been optimized over several
semesters to withstand pipetting errors and other types of mis-
takes that are common in undergraduate labs, which ensures a
high success in teaching labs that have students with various
levels of lab experience.
The most challenging experiment is most likely the isola-
tion of genomic DNA from canavanine resistant cells (Lab 3)
to confirm that gene editing has occurred. Isolating genomic
DNA from yeast cells is time-consuming and labor-intensive
because they have a strong cell wall that is difficult to lyse.
After trying different methods and kits, we settled on a yeast
DNA isolation kit made by Thermo Scientific because it was
the easiest to use and consistently yielded high quality geno-
mic DNA. However, in a class of 20 students it can take about
90 minutes for all groups to successfully isolate DNA. In addi-
tion, there were multiple instances of downtime lasting
around 10 minutes which can be challenging in a classroom
setting. We typically used this downtime to work on exercises
that supplement the lecture material and go over future steps
of the protocol to minimize mistakes. To reduce the amount
of time used to isolate genomic DNA, instructors could
employ colony PCR for this step of the lab module. We tried
multiple colony PCR protocols and found that even though
colony PCR could be used successfully to amplify the CAN1
gene, the results were not consistent, which is not ideal for a
teaching lab. Thus, we have decided to use a more laborious
protocol to isolate genomic DNA to maximize the success
rate. If pressed for time, the genomic DNA isolation step can
be eliminated and the students can be directly given the
sequence from previous canavanine editing labs (3-week ver-
sion of the module).
If instructors would like to incorporate a transformation
experiment, an additional lab could be added at the begin-
ning of this module that would entail transforming the CAN1
gRNA plasmid into Cas9 cells. This experiment would provide
an opportunity to discuss the importance of having both com-
ponents of the CRISPR/Cas9 genome editing system (the Cas9
nuclease and gRNA) in one cell so that a targeted modifica-
tion can be generated. The isolated CAN1 gRNA plasmid
7

could either be provided to the students or they could isolate
the plasmid from bacterial cells to gain experience in the
miniprep procedure. We chose not to utilize this strategy at
Suffolk because these techniques are introduced in previous
labs during the semester.
In conclusion, this gene editing lab module further high-
lights the potential of using yeast to teach CRISPR-associated
concepts in undergraduate labs. 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 mod-
ule can show students how the CRISPR/Cas9 system can gener-
ate random frameshift mutations with NHEJ, a different lab
module [15] 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 [19] that will allow students to develop a
hypothesis and to test it by designing their own gRNAs and
carrying out CRISPR. We have started designing a new set of
labs that will allow students to target various DNA repair
genes in yeast using different gRNAs. This strategy would fur-
ther enhance their knowledge about CRISPR and give them
exposure to real-world research.
Acknowledgments
We would like to thank the undergraduate students that took
BIO L274 at Suffolk University and participated in the assess-
ment exercises associated with this lab module. We thank the
various work-study students that helped in preparing reagents
and gave insightful comments about the lab manuals. We also
thank the Suffolk University Biology Department and College
of Arts and Sciences for their support of this project.
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