CRISPR-Cas9 Lab

Genetics 545

Lab Topic: This lesson focuses on CRISPR, a newer genome engineering technique. We will use this
technology in yeast to “knockout”, or remove, a gene called ADE2.

Learning Objectives:
After completing this lab, students should be able to:
1) Explain how CRISPR works and identify different components of the genetic modification
process
2) Illustrate and assemble the pieces of the CRISPR-Cas9 complex correctly in a two-dimensional
model
3) Identify a gene of interest in your organism of interest and design two guide RNAs to be used
for CRISPR
4) Apply the concepts by performing CRISPR in yeast

Introduction:
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a family of sequences in
bacteria that contain pieces of DNA, or “novel spacers” from viruses that have attacked the bacteria.
Think of this as the bacteria collecting “mementos” to remember its enemies in order to protect itself
in the future, thereby a form of acquired immunity. Using a Cas (CRISPR-associated) protein, bacteria
can recognize and cut the exogenous DNA to form the CRISPR array in its genome. If that virus
invades again, transcription of the CRISPR array occurs and CasII, a specific Cas protein, cleaves the
RNA into crRNAs, or CRISPR RNAs. The crRNAs bind to CasIII, another specific Cas protein, to form the
Cas-crRNA complex that can recognize the new incoming viral DNA and inactivate it. Below is an
image of how this works in its native form:

Source: Wikipedia
Scientists have developed a way to utilize this system in eukaryotes as a way of modifying the
genome. This simplified system requires three important pieces: a double stranded DNA of sequence
that contains a protospacer adjacent motif (PAM) sequence of NGG, with “N” being any nucleotide; a
synthesized guide RNA (gRNA) that matches to a sequence directly upstream of the PAM sequence;
and a Cas9 protein. When all three pieces form a complex, this will allow Cas9 to cut our DNA at a
precise location three base pairs upstream of the PAM sequence. This is illustrated in the image
below:

Source: University College London

To “knockout” a piece of DNA, one would need to develop gRNAs for two locations in the genome to
cut out a piece of DNA in between. If one is interested in inserting DNA instead, in addition to
developing two gRNAs, you would also add your DNA of interest that has homology to regions of DNA
that lie near the outside of the PAM sequence in order for the piece to recombine with the native
DNA.

In this lab, we will be performing CRISPR-Cas9 in yeast to knockout a gene called ADE2, which
encodes for phosphoribosylaminoimidazole carboxylase, or catalyzes a step in the ‘de novo’ purine
nucleotide biosynthetic pathway. When knocked out, this would cause red pigment to accumulate in
mutant colonies that are deprived of adenine.

***If there is a time constraint due to lab structure, can incorporate Parts 1 and 2 during
incubations steps 12 and 13 in Part 3.****

Part 1a: What do you know about CRISPR before this lesson? Instructor will facilitate a short
conversation with the class about what they know about CRISPR, if they have heard about it before or
have even used it.
Part 1b: CRISPR and Physical model. In this section, we will use a 2-dimensional model to go through
the steps of CRISPR-Cas9 in pairs. Each pair will receive three things: 2 envelopes and a paper with
Cas9 protein on it. Instructor will walk all the students through each step together.
1. Open the envelope with the number #1 on the front. Take out the three slips of paper in the
envelope. Two of the pieces are DNA, and the other is the guideRNA.
2. Highlight the PAM sequence on one of the pieces of DNA.
3. Identify the 20 bp sequence before the PAM and underline it.
4. “Design” or identify the guide RNA (gRNA). The “N” sequence is specific to create the loop
structure needed to make it a gRNA.
5. Assemble the Cas9-gRNA complex with all four pieces of paper together.
6. Dock the Cas9-gRNA complex on the DNA.
7. Identify the cut site on the DNA 3 bp before the PAM sequence. Call the instructor over to
verify your model is correct. If the model is indeed correct, they will perform the task of the
Cas9, cutting the DNA at the correct location.
8. Repeat steps 1-7 with envelope 2.
9. Come together as a class and discuss what you learned. How do the pieces needed for CRISPR
interact? What does each piece do?

Part 2: Developing a gRNA for your gene of interest. In this part of the lesson, you are asked to
develop a gRNA based on the previous activity we have done. The end goal is to implement what we
learned in Part 1 and apply it to something of interest to you. This can be done in pairs. The instructor
will walk around the class and answer questions.
1. Choose an organism of interest from the list below and follow the link to go to their
organism’s genome browser.
 Yeast (Saccharomyces cerevisiae)- https://www.yeastgenome.org
 Maize (Zea mays)- https://www.maizegdb.org
 Human (Homo sapiens)- http://useast.ensembl.org/Homo_sapiens/Info/Index
 Flies (Drosophila melanogaster)- http://flybase.org
 Mouse (Mus musculus)- http://useast.ensembl.org/Mus_musculus/Info/Index
 Worm (Caenorhabditis elegans)- http://www.wormbase.org/#012-34-5
 Zebrafish (Danio rerio) - http://useast.ensembl.org/Danio_rerio/Info/Index
2. Choose a gene of interest. This can be something you study in your own labs, something
related to a disease or pathway that is important to you, or you may just like the name of the
gene. Extract its DNA sequence from the site and copy and paste it into a word document.
3. Highlight two PAM sequences found in this gene.
4. Underline 20 bp upstream of each PAM sequence.
5. Draw a diagram including your two gRNAs you developed. Please include the Cas9 protein,
both strands of the DNA sequence, highlighted PAM sequence and gRNAs. Do not worry about
what the sequence is for the hairpin loop for the gRNA and use “N”s.
6. Propose an experiment that can be done with your gRNA. Would you create a knockout or
insert something? What do you expect that to do/find? Summarize it in 3-5 sentences.
7. Submit this word document via Canvas as either .docx or .pdf.
* Instructor notes: Facilitate a discussion as a class what we learned from this activity
afterwards. What did you choose and why? Make sure students see the value of applying
what we learned in the introduction and Part 1.
Part 3: Transformation with the pXIPHOS-ade2 construct. pXIPHOS-ade2 is a yeast shuttle plasmid
that contains an ARS, a dominant drug-resistance marker (NAT), a gene for the constitutive
expression of Cas9, a gene for the expression of a sgRNA targeting ADE2, and a donor DNA template.
ADE2 encodes for Phosphoribosylaminoimidazole carboxylase. It catalyzes a step in the 'de novo'
purine nucleotide biosynthetic pathway and a red pigment accumulates in mutant cells deprived of
adenine. All the CRISPR/Cas9 components are on one plasmid, so all you need to do is perform a
yeast transformation, recover, and select for NAT resistance. Most resistant colonies should also be
pink and unable to make purines. Steps 1-3 will have already been done for you due to time. They are
illustrated here for your general knowledge. Make sure to follow the guidelines of what to include in
your lab notebook outlined in the class syllabus. The instructor will walk around the classroom and
assist with any questions or help.

COMPLETED BEFORE START OF LAB

1. Inoculate 3mL of culture in a glass test tube for overnight growth on the room temperature
wheel.
2. The next morning, inoculate 50 mL of yeast extract peptone dextrose (YPD) in a 250-mL
Erlenmeyer flask with 1.5 mL of overnight culture and shake at room temperature for four
hours at 250 rpm. YPD is a complete medium for yeast growth.
3. After 4 hours, check the OD595. This will tell us the growth density of the yeast. If between
0.75 and 1.0, continue to step 4. If not, continue shaking.

START HERE
4. Spin down culture at 3000RPM for five minutes.
5. Remove supernatant and re-suspend cells in 25 mL of distilled water.
6. Spin down culture at 3000 RPM for five minutes.
7. Remove supernatant, add 950 μL of 100mM lithium acetate and re-suspend. Lithium acetate
is used to permeabilize the cell wall of yeast.
8. Aliquot out 100 μL of cell suspension into Eppendorf tubes (one per transformation reaction
or control).
9. Spin at max speed in centrifuge for 1 minute, then remove supernatant.
10. Add in this order:
a. 240 μL 50% PEG-4000 (used as a precipitant in DNA isolation)
b. 35 μL 1 M lithium acetate
c. 70 μL DNA solution (200-500ng plasmid DNA)
d. 5 μL single stranded salmon sperm DNA (10 mg/mL) (this protects the plasmid from
degradation and helps the plasmid get into the yeast cell because the single stranded
DNA preferably binds to the cell wall)
11. “Washboard” tubes to re-suspend pellet in transformation mixture.
12. Incubate at 30 degrees Celsius for 30 minutes.
13. Incubate at 37 degrees Celsius for 42 minutes to heat shock the sample.
14. After heat shock, immediately add 600 μL YPD or distilled water.
15. Add transformation suspension to each of the three YPD plates: 1 plate will have 100 μL, 1 will
have 200 μL and 1 will have 300 μL. Spread with glass beads.
16. Remove beads and incubate at room temperature upside down. Grow for 16-24 hours at 30
degrees Celsius.
 17. Replica plate to new YPD plates with 100 μg/mL Nourseothricin (NAT). Grow for two days at
30 degrees Celsius.
18. If successful, colonies should appear after two days. Colonies should be pink in color. Leaving
the plates out at room temperature for a couple days should enhance the color. Some
colonies may appear to be a mixture of pink and white, suggesting that the CRISPR knockout
occurred late in the formation of the colony.
19. Take a picture of your plate and reflect on your experiment in your lab notebook. Do you have
pink colonies? Are they mixed in color? Is there anything you could do to improve the
outcome of your experiment?
20. Individual colonies can be picked and streaked to YPD plates.
21. The CRISPR plasmid will quickly be lost upon growth under non-selection.

At the beginning of the next class, we will have a short discussion over the lab material and ask
what results they found. Were they successful in their experiment?

***Instructors Notes:
Activities:
 2-dimensional model (evaluates LOs 1 & 2)
 Develop your own gRNA activity (evaluates LOs 1 & 3)
 Lab utilizing CRISPR knocking out ADE2 in yeast (evaluates LOs 1 & 4)
Assessments:
 gRNA assignment turned in via Canvas (evaluates LOs 2 & 3)
 Lab Notebook write up (evaluates LOs 1 & 4)