This document summarizes an experiment aiming to knockout the CTNNB1 gene in mouse embryonic stem cells using CRISPR-Cas9 genome editing. It describes designing a guide RNA targeting exon 10 of CTNNB1, cloning it into a vector, transforming E. coli, and testing primers for detecting knockout via PCR and qPCR. Prior studies showed CTNNB1 knockout embryos had defects in ectoderm and mesoderm development. The authors hypothesized knocking out CTNNB1 in stem cells would produce inviable embryos, similar to prior findings.

1. Reduction in
Expression of
CTNNB1 in Mouse
Embryonic Stem Cells
via CRISPR-Cas9
Genome Editing
The University of Colorado, Denver
BIOL 4125
5/10/2016
Samuel Altman

2. Introduction and Background
CRISPR-Cas9 genome editing is a potentially revolutionary new tool for the
manipulation of genomic material across all species. The term CRISPR is an acronym that stands
for clustered regularly interspaced short palindromic repeats. These repeats were first described
by Japanese researchers who discovered them in Escherichia coli in 1987 (Doudna and
Charpentier 2014). Since their discovery, CRISPRs have been found in many other bacteria. It
was proposed that CRISPRs were part of the bacterial adaptive immune system in which
antisense RNA fragments are generated from the CRISPRs integrated in the bacteria’s genome in
response to the presence of viral double stranded DNA. The RNA fragments associate with a Cas
protein which is an endonuclease that can initiate double stranded DNA breaks. The crRNA
(CRISPR RNA) and Cas protein complex then locate the complementary sequence of the
invading viral DNA, bind to the recognition site, and the Cas protein cleaves the double stranded
DNA, effectively destroying the viral DNA invader. Strands of the cleaved viral DNA are then
integrated into the host’s genome as a new CRISPR and a defense against a repeated invasion
from that virus.
The Cas-9 protein is just one variant within the Cas protein family and a particularly
effective dual RNA guided DNA endonuclease (Doudna and Charpentier 2014). The CRISPR-
Cas9 complex uses a duplex of tracrRNA and crRNA to direct DNA cleavage at a designated site
(Doudna and Charpentier 2014). The Cas9 protein contains an HNH domain and a RuvC-like
domain that can be mutated in order to alter the functionality of the Cas9 protein (Doudna and
Charpentier 2014). By mutating one of these domains, a variant Cas9 protein is produced that
will cleave only a single strand of DNA whereas mutation of both domains eliminates the Cas9’s
endonuclease ability but allows it to be an effective and specific RNA guided DNA binding
protein (Doudna and Charpentier 2014). The variability of the Cas9 protein provides numerous
means to manipulate genomic DNA for therapeutic advances such as: DNA deletion, insertion,
replacement, modification, and labeling, as well as transcription modulation and RNA targeting
(Doudna and Charpentier 2014).
For this experiment, neither the HNH nor the RuvC-like domains were mutated which
allowed for the harnessing of the CRISPR-Cas9’s double strand DNA break mechanism to
potentially knockout a gene of interest. After a double stranded DNA break is made, cells have
two mechanisms to repair the genetic damage, non-homologous end joining (NHEJ) and
homology directed repair (HDR). In the case of NHEJ, DNA damage is repaired by ligating the
ends of the broken DNA fragments together after accessory factors bind the ends of the
fragments and extend the strands after they are partially resected (Wyatt and Ramsden 2015).
Through this process, the double strand break is repaired and indel errors are introduced into the
sequence which have about a two thirds chance of causing a frameshift mutation which can
result in a potentially nonfunctional protein (Wyatt and Ramsden 2015).
Homology directed repair (HDR) begins with a resection of the 5’ ended DNA strand to
create a 3’ overhang (Cortez 2015). The invasive strand can then displace one strand of the

3. homologous DNA duplex and pair with the other which creates a displacement loop (Cortez
2015). At this point, repair can be completed by the Classical Double-Strand Break Repair
Pathway (DSBR) or the Synthesis-Dependent Strand-Annealing Pathway (SDSA) (Cortez 2015).
In the classical DSBR pathway, the 3’ ends of the DNA fragments annex an intact singles
stranded homologous template then serve as primers for DNA repair synthesis which leads to the
formation of double Holliday junctions (dHJs); the individual Holliday junctions are then
resolved via cleavage (Cortez 2015). Distinct from DSBR, after strand invasion and
displacement loop formation in SDSA, the newly produced portion of the invasive strand is
removed from the template and reverted to the processed end of the non-invading strand at the
other double strand break end (Cortez 2015). The 3’ end of the non-invasive strand is then
extended and ligated to fill the remaining gap which completes the SDSA repair process (Cortez
2015). The DSBR pathway often leads to crossover while the SDSA pathway is conservative and
does not result in crossover (Cortez 2015). For this experiment, NHEJ repair was utilized after
genome editing with the CRISPR-Cas9 complex to knockout the CTNNB1 (Beta-catenin) from
mouse embryonic stem cells.
Beta-catenin is a protein that serves, in particular, two critical functions, regulating the
coordination of cell to cell adhesion and gene transcription. Beta-catenin’s role as a transcription
co-activator is primarily detailed in the Wnt signaling pathway. Beta-catenin serves as a signal
for cells to proliferate and is regulated by phosphorylation via GSK3-beta. Singaling via the Wnt
pathway is one of the fundamental processes that drives cellular proliferation, cell polarity, and
cell fate determination in early embryonic development (MacDonald et. al 2009). Mutations in
the Wnt pathway are known to be associated with birth defects such as osteogenesis imperfecta
and tetra-amelia and are related to multiple types of cancer (MacDonald et. al 2009).
Beta-catenin also serves an important role in the cadherin-catenin adhesion complex.
Catenins facilitate the connection of cadherins with actin microfilaments and are part of higher
order protein structure in which cadherins are associated with other cytoplasmic and
transmembrane proteins (Hoschuetzky et. al 1994). Beta-catenin knockout experiments have
been conducted before to determine its function within animal systems. One such study utilized
mouse embryonic stem cells in which Beta-catenin was knocked out. In knockout embryos,
seven days post coitum, the development of the embryonic ectoderm was affected (Haegel et. al
1995). Cells dislodged from the ectodermal layer and were found to be dispersed throughout the
proamniotic cavity suggesting Beta-catenin’s role in cadherin-catenin complexes is particularly
important for the development of the ectodermal layer (Haegel et. al 1995). Additionally,
knockout embryos did not develop the mesodermal cell layer (Haegel et. al 1995).
Utilizing the information from prior studies it was hypothesized that knocking out Beta-
catenin from mouse embryonic stem cells via the CRISPR-Cas9 genome editing mechanism
would result in inviable embryos similar to those found in the Haegel experiment if cells were
allowed to mature. In this experiment, mouse embryonic stem cells were not allowed to mature
beyond the replicative stages necessary to colonize transfected cells for use in T7 and qPCR
assays.

4. Methods
Gene Block and PCR Primers Design
Gene Block Synthesis: Using the E-Crisp website, three 23 base pair genomic sites on three
different axons of the gene CTNNB1 (Beta Catenin) were chosen as potential guide RNA target
sequences. Using NCBI BLAST, sequences were queried to ensure that the selected sequences
were not duplicated anywhere else in the genome. The sequence which targeted exon 10 of the
CTNNB1 gene in mouse embryonic stem cells was chosen to be the guide RNA sequence. 19
base pairs of the target sequence were incorporated into the gene block design of the Church Lab
protocol (Figure 1). The 455 base pair gene block, including the U6 promoter, target sequence,
guide RNA scaffold, and termination signal, was synthesized as a gBlock and ordered from IDT
before being cloned into the pCR-BLUNT II-TOPO vector (Figure 2).
PCR Primers Design: The guide RNA target sequence obtained from E-CRISP was blasted
using a nucleotide blast optimized for highly similar sequences in the mouse genomic and
transcript database. After ensuring that the target sequence corresponded with the CTNNB1
gene, 1000 base pairs were added to both sides of 19 base pair fragment by changing the region
of the reference sequence. The new sequence was blasted using a primer blast to generate a list
of viable PCR primers (Figure 3). Primer 4 was selected as it captured the target sequence and
provided ample sequence length that flanked both ends of the target. Additionally, predesigned
qPCR primers for mouse CTNNB1 were selected for use in the qPCR assay and ordered from
IDT.
Genomic DNA Isolation: Neuro-2a cells from mice were trypsinized and scraped from the
growth surface of the plate. They were then centrifuged at 500 x g for two minutes before being
lysed with the addition of genomic lysis buffer. The mixture was transferred to a Zymo-Spin
column in a collection tube and centrifuged at 10,000 x g for one minute. The flow through was
discarded and 200 μl of DNA pre-wash buffer was added to the spin column, after which it was
centrifuged at 10,000 x g for one minute. 500 μl of genomic DNA wash buffer was then added to
the spin column, followed by centrifugation at 10,000 x g for one minute. The spin column was
transferred to a clean microcentrifuge tube after which 50 μl of DNA elution buffer was added.
The column was incubated for three minutes at room temperature, followed by centrifugation at
21,000 x g for thirty seconds to elute the genomic DNA. The DNA was quantified and was found
to be at a concentration of 30 ng/μl.

5. PCR PRIMERS ANALYSIS
Both the forward and reverse PCR primers were diluted to 200ng/μl before being utilized in the
PCR reaction (Table 1).
Reagent Sample (μl) Negative Control (μl)
gDNA (30ng/ μl) 3.34 0
Forward Primer (200ng/ μl) 1 1
Reverse Primer (200ng/ μl) 1 1
PCR Master Mix 12.5 12.5
Water 7.16 10.5
Total 25 25
Table 1: PCR reaction set-up for PCR primer test on mouse genomic DNA from neuro-2a cells.
The sample and negative control were loaded into a thermal cycler and underwent the PCR
reaction using the conditions in Table 2.
Step Temperature
(˚C)
Time Cycles
Initial
Denaturation
95 10 min 1
Denaturation 95 30 sec X35
Annealing 55 30 sec
Extension 72 1 min
Additional
Extension
72 5 min 1
Hold 4 HOLD --
Table 2: Thermal cycler conditions for the PCR reaction testing the efficacy of the PCR primers.
8μl of each PCR product was mixed with 2μl of loading dye before being run on a gel (Figure 4).
RNA Isolation and qPCR Primer Test
RNA Isolation: A plate containing mouse neuro-2a cells was obtained and the media was
pipetted off. 500μl of Trizol was added to the plate and allowed to incubate for 5 minutes. The
cells were washed to the bottom of the plate by pipetting the mixture on the plate which was
angled. The mixture was then transferred into a microcentrifuge tube and centrifuged at 12,000 x
g for one minute. The supernatant was transferred into a clean microcentrifuge tube and 500μl of
ethanol was added and mixed thoroughly. The mixture was then placed into a Zymo-spin column
in a collection tube and centrifuged for 30 seconds at 12,000 x g. The flow through was
discarded and the column was placed into a new tube. 400µl of RNA PreWash was added to the
column which was then centrifuged at 12,000 x g for 30 seconds. This wash step was repeated an
additional time after which the flow through was discarded. 700µl of RNA Wash Buffer was
then added and centrifuged for 2 minutes at 12,000 g to ensure the wash buffer was completely

6. removed. The column was then transferred into an RNase-free tube. The RNA was eluted by
adding 50µl of DNase/RNase free water to the spin column which was then centrifuged at
12,000 x g for one minute. The RNA was then quantified and the concentration was found to be
116 ng/µl. 5 µl of the RNA solution was mixed with 1 µl of loading dye and loaded into and run
on a gel (Figure 5).
cDNA synthesis: A 2X reverse transcriptase master mix was created by mixing 2μl of 10X R.T
buffer with 0.8μl of 25X dNTP Mix (100mM), 2μl of 10X R.T random primers, 1μl of
MultiScribe reverse transcriptase, and 4.2μl of nuclease free water. The 10μl of 2X R.T master
mix was added to 10 μl of RNA (116 ng/μl) before the reverse transcriptase reaction was
initiated using the following thermal cycler conditions (Table 3).
Step 1 Step 2 Step 3 Step 4
Temperature
(°C)
25 37 85 4
Time 10 min 120 min 5 miin Hold
Table 3: Reverse transcriptase reaction conditions for cDNA synthesis.
The prepared cDNA was diluted before being utilized in qPCR. To dilute the cDNA sample to
8.89 ng/μl, 102.5 μl of DEPC water was added to 20 μl of the cDNA solution.
Quantitative PCR: To prepare the CTNNB1 PrimeTime probe, 500μl of sterile TE buffer was
added to the probe which diluted it to 20x. The GAPDH qPCR probe was pre-concentrated at
40x. GAPDH was chosen as a positive control for the qPCR assay. Master mixes for β-catenin
and GAPDH were made according the process detailed in Table 4. After the master mixes were
prepared, 19 µl of the GAPDH master mix was added into wells A-C in columns one and two
and19μl of the CTNNB1 master mix was added to wells D-F in wells one and two. 6 µl of
diluted cDNA was loaded into all twelve wells.
β-catenin GAPDH
Reagent Volume
(µl)
Replicate X3.2 Reagent Volume
(µl)
Replicate X3.2
2X
PrimeTime
master mix
12.50 40 µl 2X
PrimeTime
master mix
12.50 40 µl
20X β-
catenin
probe
1.25 4.0 µl 40X
GAPDH
Probe Mix
0.625 2.0 µl
Water 5.63 18 µl Water 6.255 20 µl
Diluted
cDNA
*6 − Diluted
cDNA
*6 −
Total 25.0 25.0
Table 4: β-catenin and GAPDH master mix protocol for sue in qPCR. Diluted cDNA was not added to the master
mix.

7. After the well plate was set up and the mixtures were deposited, the well plate was added into the
qPCR thermal cycler set to the conditions in Table 5. The qPCR thermal monitored the
fluorescence signal as amplification proceeded (Figure 6).
Temperature Time Cycles
50°C 2’ 1
95°C 10’ 1
95°C 15”
X 40
60°C 1’
Table 5: qPCR thermal cycler conditions for the assay of the CTNNB1 qPCR primer test.
Vector Cloning, Cell Transformation, and Restriction Digestion
pCR-Blunt II-TOPO Cloning: The synthesized gBlock was cloned into a pCR-Blunt II-TOPO
vector by adding 2 μl of the gBlock to 1 μl of a salt solution, 1 μl of the pCR-Blunt II-TOPO
vector and 2 μl of sterile water. This 6 μl cloning solution was mixed gently and incubated at
room temperature for five minutes before being used in transformation.
One Shot Cell Transformation: 2μl of the TOPO cloning solution were added to 25μl of
OneShot Chemically Competent E. coli cells and mixed gently. The reaction was then incubated
on ice for twenty minutes before being heat shocked at 42 degrees Celsius for thirty seconds. The
reaction was then immediately transferred to ice. 250μl of S.O.C medium was added to the cells
after which the reaction was shaken horizontally (225rpm) at 37 degrees Celsius for 45 minutes.
Cell Plating: 100μl of the OneShot cell transformation were added center of a pre-warmed LB
plate containing 50μg/ml of kanamycin. 8 glass beads were added to the plate which was then
shaken side to side and front to back in order to spread the transformation. The plate was then
inverted and incubated overnight at 37 degrees Celsius.
Colony Analysis: 4 colonies were selected from the transformation plate. Each colony was
cultured overnight in 3 mL LB broth and 15μL Kanamycin (50 μg/mL).
Blunt II-TOPO Plasmid DNA Miniprep: Bacterial cultures from the colony selection were
harvested by centrifugation at 8000rpm for two minutes after which the supernatant was
decanted. The pelleted cells were re-suspended in 250μl of re-suspension solution containing
RNase-A. 250μl of lysis solution was then added and mixed by inverting the tubes 6 times. 350μl
of neutralization solution was then added and the tubes which were inverted six more times to
mix the reagents. The mixtures were then centrifuged for five minutes at 14,000 rpm to pellet the
cell debris and chromosomal DNA after which the supernatant was decanted and added to
GeneJet spin columns. The solutions were centrifuged in the spin columns for one minute at
14,000rpm after which the flow-through was discarded. 500μl of wash solution was then added
to the columns which were spun again for one minute at 14,000 rpm with the flow-through being
discarded. The wash procedure was repeated once more followed by an additional centrifugation
for one minute at 14,000 rpm without any solution added in order to remove any residual wash
solution. The spin columns were transferred to a microcentrifuge tube after which 50μl of elution

8. buffer was added to the spin columns’ membranes. The columns were incubated at room
temperature for two minutes after which they were centrifuged for two minutes at 14,000 rpm to
extract the isolated plasmid. The samples were quantified to determine the concentration of
collected plasmid DNA (Table 6).
Sample DNA
Concentration
(ng/μL)
1 70
2 100
3 135
4 110
Table 6: Concentrations of pCR-Blunt II-TOPO plasmid DNA extracted via miniprep.
Restriction Digest: To analyze the presence and orientation of the gBlock insertion in the pCR-
Blunt II-TOPO vector, the plasmid samples were digested using the XBA1 restriction enzyme.
Restriction digest samples were prepared according to the set-up in Table 7. Samples were
incubated for five minutes at 37 degrees Celsius before being loaded into the gel.
Sample 1
(70ng/μl)
2
(100ng/μl)
3
(135ng/μl)
4
(110ng/μl)
Plasmid
DNA (μl)
14.3 10 7.4 9.1
Water (μl) 2.7 7 9.6 7.9
Restriction
Digest
Buffer (μl)
2 2 2 2
XBA1 (μl) 1.0 1.0 1.0 1.0
Total (μl) 20 20 20 20
Table 7: Restriction digest of pCR-Blunt II-TOPO plasmid samples using XBA1.
Restriction Digest Electrophoresis: 6 μl of each restriction digest sample and 1 μl of loading
buffer were loaded into separate gel wells (Figure 7).
Sequencing. Samples 1 and 4 were selected for sequencing. Sample 1 had the inverted
orientation and sample 4 had the proper orientation as indicated by the gel image in Figure 7. 2.8
μl of DNA from sample 1 (70ng/μl) was added to 6.2 μl of water. 1.8 μl of DNA from sample 4
(110 ng/μl) was added to 7.2 μl water. The mixtures were sent to Eton Biosciences for
sequencing (Figure 8).

9. Transfection of Mouse Embryonic Stem Cells Using PEI
Reagents.
 Feeder-free wild-type mouse embryonic stem cells.
 Basic cell growth medium (see reagent setup).
 Transfection medium (see reagent setup).
 Trypsin-EDTA solution, 0.05% (Gibco cat. no. 25300054).
 Opti-MEM reduced serum medium (Gibco cat. no. 31985070).
 PBS, pH 7.4 (Gibco cat. no. 10010-023).
 Gelatin solution diluted in PBS, pH 7.4.
 Polyethylenimine, linear, 25 kDa (Polysciences, Inc. cat. no. 23966).
 HEPES.
 NaCl.
 Na2HPO4.
 NaOH.
 Mammalian gene expression plasmids.
 0.4% Trypan Blue
Equipment.
 Advanced Microscopy Evos fluorescent microscope.
 5% CO2, 37°C sterile incubator.
 Tissue-culture 6-well plate.
 Tissue-culture 10 cm dish.
 0.2 μm syringe filter and syringes.
 Polypropylene microcentrifuge tubes.
 15 mL polypropylene centrifuge tube.
 Centrifuge with swing bucket rotor.
 Pasteur pipettes.
 Filter tips.
 Biological safety cabinet (BSC).
 Countess Automated Cell Counter with provided slides or hemocytometer
Basic ESC culture growth medium.
 high glucose DMEM (Gibco cat. no. 11960-044) supplemented with 15% fetal bovine
serum (HyClone cat. no. SH3007103),
 1% non-essential amino acids (Gibco cat. no. 11140050),
 1% sodium pyruvate (Gibco cat. no. 11360070),
 1% L-glutamine (Gibco cat. no. 25030081),
 1% penicillin/streptomycin (Gibco cat. no. 15140-122),
 55 μm 2-mercaptoethanol (Gibco cat. no. 21985-023) added fresh,
 1000 units/mL ESGRO (Millipore cat. no. ESG1106) added fresh.

10. ES cell transfection medium;
 high glucose DMEM supplemented with 18% fetal bovine serum,
 1.2% non-essential amino acids,
 1.2% sodium pyruvate,
 1.2% L-glutamine,
 1.2% penicillin/streptomycin,
 55 μm 2-mercaptoethanol added fresh,
 1000 units/mL ESGRO (or LIF) added fresh.
PEI Solution;
 40 μM linear PEI, 25 kDa (Polysciences, Inc. cat. no. 23966) diluted in 25 mM HEPES
buffer containing 140 mM NaCl, 1.5 mM Na2HPO4, pH to 7.05 with 5N NaOH.
Transfection: A 10 cm dish of mouse embryonic stem cells was grown in basic ES cell growth
medium. Confluency was determined by visualizing the 10 cm dish of ES cells under transmitted
light of a microscope (≈90% confluent). All reagents were warmed to room temperature before
being used. The basic ES cell growth medium was aspirated off in a hood. The Adhered ES cells
were washed with 1 mL PBS. 2 mL 0.05% Trypsin-EDTA was then added to the plate of ES
cells and incubated in the 5% CO2, 37°C sterile incubator for 5 minutes. ES cells were collected
by pipette and deposited in a 15 mL conical tube which was then centrifuged 1500 rpm for 2
minutes. The supernatant was aspirated off. ES cells were re-suspended in enough Opti-MEM
such that the Countess/hemocytometer was able to calculate an accurate cell number. 10 μl of
Trypan Blue solution was mixed with 10 μl of the re-suspended cells and mixed by pipetting the
solution up and down. Cells were counted using Countess and 1x106 cells were selected and
deposited in a microcentrifuge tube. The tube was centrifuged at 1500 rpm for two minutes after
which the supernatant was aspirated off. The ES cells were re-suspended in 300 μl of Opti-MEM
solution. 1μg of pCas9-GFP plasmid (Figure 13) and 1μg of pCR Blunt II-TOPO plasmid
containing the guide RNA was added to the re-suspended ES cells. 100 μl of PEI solution was
added to the solution containing the ES cells, plasmid DNA, and Opti-MEM solution which was
incubated for 30 minutes at room temperature, which was mixed gently every ten minutes. 2 ml
of the mouse embryonic stem cell transfection was added dropwise to a plate containing 0.1%
gelatin. A fluorescent microscope was used to monitor the efficacy of the transfection by
detecting for GFP.
Cell Splitting, gDNA Isolation, T7 Endonuclease Cutting, and qPCR
Splitting Cells: Media was aspirated off of the plate containing transfected mouse embryonic
stem cells and the plate was washed with 1 mL PBS which was aspirated off after the plate was
gently rocked. 2 mL Trypsin was then added to the plate which was then incubated in the 5%
CO2, 37°C sterile incubator for 5 minutes. The mixture was pipette into a microcentrifuge tube
and centrifuged for 3 minutes at 1500 x g. The trypsin was then pipetted off and the cells were
re-suspended in 1 ml of embryonic stem cell media. 500 μl of the cells was added to a fresh plate
with gelatin and embryonic stem cell media; the remaining cells were used to isolate genomic
DNA.

11. Genomic DNA Isolation: 500μl of transfected mouse embryonic stem cell were trypsinized and
scraped from the growth surface of the plate. They were then centrifuged at 500 x g for two
minutes before being lysed with the addition of genomic lysis buffer. The mixture was
transferred to a Zymo-Spin column in a collection tube and centrifuged at 10,000 x g for one
minute. The flow through was discarded and 200 μl of DNA pre-wash buffer was added to the
spin column, after which it was centrifuged at 10,000 x g for one minute. 500 μl of genomic
DNA wash buffer was then added to the spin column, followed by centrifugation at 10,000 x g
for one minute. The spin column was transferred to a clean microcentrifuge tube after which 50
μl of DNA elution buffer was added. The column was incubated for three minutes at room
temperature, followed by centrifugation at 21,000 x g for thirty seconds to elute the genomic
DNA. The genomic DNA was quantified and found to be concentrated at 5 ng/ μl. The genomic
DNA was used to run PCR according to the set-up in Table 8. 5 μl of PCR product and 1 loading
dye were mixed and add to a gel before being run (Figure 9).
Reagent Sample (μl)
gDNA (5ng/ μl) 12
Forward Primer (200ng/ μl) 1
Reverse Primer (200ng/ μl) 1
PCR Master Mix 12.5
Water 0
Total 26.5
Table 8: PCR reaction set-up for amplification of genomic DNA extracted from transfected mouse embryonic stem
cells.
T7 Endonuclease: 5 μl of DNA from the PCR reaction was added to 2 μl of 10XNEBuffer 2 and
12 μl of nuclease free water. The PCR products were annealed in a thermocycler using the
conditions in Table 9.
Step Temperature Ramp
Rate
Time
Initial
Denaturation
95 ˚C 30 s
Annealing 95-85 ˚C -2 ˚C /s 5 s
85-25 ˚C -0.1 ˚C/s 10 s
Final
Extension
72 ˚C 5 m
Table 9. Thermal cycling conditions for the annealing of PCR products before the addition of T7 endonuclease.
1 μl of T7 endonuclease I was added to the 19 μl mixture of annealed PCR products and
incubated for 15 minutes at 37 ˚C. The reaction was halted by the addition of 1.5 μl of 0.25 M
EDTA. The DNA fragments were eluted with 20 μl of water and 15 μl of the elution was
analyzed through gel electrophoresis (Figure 10).

12. One-Shot cells were transformed using the OneShot transformation protocol utilized previously
with 1μl of CTNNB1 plasmid extracted from the previously transformed OneShot cells. Sample
number four from the previous transformation was used in this transformation. Plasmid DNA
was extracted from these OneShot cells by utilizing the same miniprep process as previously
performed. Quantification of the isolated plasmid DNA revealed that the DNA concentration of
the extracted sample was 50ng/μl. Mouse embryonic stem cells were transfected with the
extracted pCR Blunt II-TOPO plasmid containing the gRNA and the pCAS9-GFP plasmid
according to the same transfection protocol that was previously used. The transfected cells were
split once more using the cell splitting protocol and genomic DNA was isolated from half of the
split cells according to the genomic DNA isolation protocol used previously. The genomic DNA
was quantified and its concentration was found to be 5ng/μl. A full PCR reaction was performed
according to the set-up in Table 10. 5 μl of PCR product and 1 loading dye were mixed and add
to a gel before being run (Figure 11).
Reagent Sample (μl)
gDNA (5ng/ μl) 24
Forward Primer (200ng/ μl) 2
Reverse Primer (200ng/ μl) 2
PCR Master Mix 25
Water 0
Total 53
Table 10: PCR reaction set-up for amplification of genomic DNA extracted from transfected mouse embryonic stem
cells.
RNA Isolation: A plate containing transfected mouse embryonic stem cells was obtained and
the media was pipetted off. 500μl of Trizol was added to the plate and allowed to incubate for 5
minutes. The cells were washed to the bottom of the plate by pipetting the mixture on the plate
which was angled. The mixture was then transferred into a microcentrifuge tube and centrifuged
at 12,000 x g for one minute. The supernatant was transferred into a clean microcentrifuge tube
and 500μl of ethanol was added and mixed thoroughly. The mixture was then placed into a
Zymo-spin column in a collection tube and centrifuged for 30 seconds at 12,000 x g. The flow
through was discarded and the column was placed into a new tube. 400µl of RNA PreWash was
added to the column which was then centrifuged at 12,000 x g for 30 seconds. This wash step
was repeated an additional time after which the flow through was discarded. 700µl of RNA
Wash Buffer was then added and centrifuged for 2 minutes at 12,000 g to ensure the wash buffer
was completely removed. The column was then transferred into an RNase-free tube. The RNA
was eluted by adding 50µl of DNase/RNase free water to the spin column which was then
centrifuged at 12,000 x g for one minute. The RNA was then quantified and the concentration
was found to be 120 ng/µl.
cDNA synthesis: A 2X reverse transcriptase master mix was created by mixing 2μl of 10X R.T
buffer with 0.8μl of 25X dNTP Mix (100mM), 2μl of 10X R.T random primers, 1μl of
MultiScribe reverse transcriptase, and 4.2μl of nuclease free water. The 10μl of 2X R.T master

13. mix was added to 8.3 μl of RNA (120 ng/μl) before the reverse transcriptase reaction was
initiated using the following thermal cycler conditions (Table 11).
Step 1 Step 2 Step 3 Step 4
Temperature
(°C)
25 37 85 4
Time 10 min 120 min 5 miin Hold
Table 11: Reverse transcriptase reaction conditions for cDNA synthesis.
The prepared cDNA was diluted before being utilized in qPCR. To dilute the cDNA sample to
8.89 ng/μl, 102.5 μl of DEPC water was added to 20 μl of the cDNA solution.
Quantitative PCR: Master mixes for β-catenin and GAPDH were made according the process
detailed in Table 12. After the master mixes were prepared, 19 µl of the GAPDH master mix was
added into wells A-C in columns one and two and 19μl of the CTNNB1 master mix was added to
wells D-F in wells one and two. 6 µl of diluted cDNA was loaded into all twelve wells.
β-catenin GAPDH
Reagent Volume
(µl)
Replicate X6.2 Reagent Volume
(µl)
Replicate X6.2
2X
PrimeTime
master mix
12.50 77.5 µl 2X
PrimeTime
master mix
12.50 77.5 µl
20X β-
catenin
probe
1.25 7.75 µl 40X
GAPDH
Probe Mix
0.625 3.875 µl
Water 5.63 34.9 µl Water 6.255 38.781 µl
Diluted
cDNA
*6 − Diluted
cDNA
*6 −
Total 25.0 25.0
Table 12: β-catenin and GAPDH master mix protocol for sue in qPCR. Diluted cDNA was not added to the master
mix.
After the well plate was set up and the mixtures were deposited, the well plate was added into the
qPCR thermal cycler set to the conditions in Table 13. The qPCR thermal monitored the
fluorescence signal as amplification proceeded (Figure 12).
Temperature Time Cycles
50°C 2’ 1
95°C 10’ 1
95°C 15”
X 40
60°C 1’
Table 13: qPCR thermal cycler conditions for the assay ofthe CTNNB1 qPCR primer test.

14. Results
Figure 1: Animated image of the gBlock design from the Church Lab protocol.

15. Figure 2: pCR Blunt II TOPO vector containing the 455bp gBlock. EcoR1 restriction sites flanked the TOPO sites
where the gBlock inserted into the vector.

16. Figure 3: Top image: A graphical view of potential PCR primer pairs for CTNNB1 generated by PrimerBlast.
Bottom image: Information and characteristics of PCR primer pair 4 which was selected to be utilized in the
CRISPR/Cas9 genome editing experiment.

17. Figure 4: A gel image of the PCR primer test.The expected band size generated by amplification using primer pair 4
was 798 base pairs.
Figure 5: A gel image of RNA extracted from mouse neuro-2a cells. Two bands were expected, the larger
corresponding to the 28S band and the smaller corresponding to the 18S band.

18. Figure 6: The amplification plot from the qPCR of GAPDH and B-Catenin qPCR primer test.The highlighted
column contains the wells shown in the plot. The triangles in the three GAPDH wells indicates that the Ct value was
unable to be determined for the GAPDH wells. The Ct values for the B-Catenin wells highlighted in pink indicate
that amplification was successfuland the consistency between the Ct values indicates that the samples were pipetted
consistently.
Figure 7: A gel image of the XBA1 restriction digest of the pCR Blunt II-TOPO vector. A band corresponding to the
proper orientation of the insert was expected at 80 base pairs. A band corresponding to the improper orientation of
the insert was expected at 500 base pairs. Samples one and two presented an insert with the improper orientation and
samples three and four presented an insert with the proper orientation.

19. Figure 8: Top image: Sequencing data from Eton Biosciences of the improper insert orientation in the pCR Blunt II-
TOPO vector obtained from sample one of the restriction digest (Figure 7). Bottom image Sequencing data from
Eton Biosciences of the proper insert orientation in the pCR Blunt II-TOPO vectorobtained from sample four of the
restriction digest (Figure 7). Inserts were flanked by Xba1 restriction sites.
Figure 9: Gel image of PCR products from genomic DNA extracted from transfected mouse embryonic stem cells.
The expected band size of the DNA fragment that was amplified by the PCR primers was 800 base pairs.
Figure 10: Gel image of the T7 assay performed on genomic DNA extracted from transfected mouse embryonic
stem cells. Two bands were expected. One band at 800 base pairs was expected which would correspond to intact

20. DNA fragments. The other band was expected at around 400 base pairs which would correspond to cleaved DNA
fragments by the T7 endonuclease.
Figure 11: Gel image of PCR products from genomic DNA extracted from transfected mouse embryonic stemcells.
The expected band size of the DNA fragment that was amplified by the PCR primers was 800 base pairs.

21. Figure 12: The top amplification plot corresponds to WT cDNA wells. GAPDH was used as a positive control for
the qPCR assay.The bottom amplification plot corresponds to cDNA generated from mouse embryonic stemcells
edited via the CRISPR/CAS9 genome editing procedure intended to knockdown/out expression of CTNNB1 (Beta-
catenin).

22. Figure 13: pCAS9-GFP Plasmid Map

23. Discussion
Before the editing of mouse genomic DNA could be undertaken, the PCR primers
designed to amplify the region of CTNNB1 (Beta-catenin) where the Cas9 endonuclease would
create the double strand break had to be tested. To test the efficacy of the PCR primers, the
primers were tested on genomic DNA extracted from mouse neuro-2a cells. The expected band
size for the amplification fragment of the gDNA was 800 base pairs which was evident in the gel
run on the primer test shown in Figure 4.
The qPCR primers ordered from IDT also needed to be tested to ensure that they would
work properly when utilized for the detection of CTNNB1 in edited cells. RNA was isolated
from mouse neuro-2a cells and purified before being run on a gel shown in Figure 5. The
presence of the 28S and 18S ribosomal bands indicated that the RNA was isolated properly and
remained intact throughout the process. The isolated RNA was then used to synthesize cDNA for
use in a trial run of qPCR as shown in Figure 6. The Ct values for the CTNNB1 wells indicate
that amplification successfully occurred and their small variance indicates that the samples were
pipetted consistently. The average Ct value for the three CTNNB1 wells was 23.783. The
GAPDH wells, which were used as a positive control since GAPDH is known to be expressed in
mouse neuro-2a cells, had undetermined Ct values. A possible explanation for this is the qPCR
probe did not work in mouse neuro-2a cells since the probe was able to detect GAPDH
expression in other cell lines.
With the PCR and qPCR primers producing positive test results, the next step was to test
the efficacy of cloning the gBlock which contained the guide RNA sequence into a pCR Blunt-II
Topo vector as shown in Figure 2. To test for the presence and orientation of the gBlock insert,
the cloned pCR Blunt II-TOPO vector was isolated from the OneShot cells that were replicating
the plasmid and was digested using XBA1. XBA1 was chosen as the restriction enzyme because
it had two restriction sites, one outside of the insert and one within. The location of these
restriction sites would help identify the orientation of the insert as the smaller fragment, about 80
base pairs, corresponded to the proper orientation and the larger fragment, about 500 base pairs,
corresponded to the improper orientation. The gel image in Figure 7 details the results of the
restriction digest and the sequencing data from Eton Biosciences (Figure 8) confirms that
samples one and two contained the improperly oriented insert and samples three and four
contained the properly oriented insert.
With the completion of these tests, transfection of mouse embryonic stem cells with the
pCR Blunt II-TOPO vector containing the gBlock insert and the pCas9-GFP vectors could
commence. The pCas9-GFP vector was chosen as the vector to carry the Cas9 protein because
the GFP tag could be utilized to determine which cell colonies were successfully transfected with
the vector. Since cells that uptake one plasmid have a large chance of uptaking the second
plasmid, it was assumed that colonies that were tagged with GFP had also been transfected with
the TOPO vector containing the gBlock.

24. Transfected colonies were isolated and the cells were split, half being re-plated and
allowed to replicate, the other half being used for PCR and T7 assays. The results of the PCR
amplification of transfected mouse genomic DNA are shown in Figure 9. The expected band size
was 800 base pairs which is confirmed in the gel image. The isolated genomic DNA was then
used in the T7 assay. Since the PCR primers used to amplify the DNA were approximately
symmetric about the site of the double strand break initiated by the Cas9 protein, it was expected
that the T7 endonuclease would produce two bands when the assay was run on a gel. The 800
base pair band would correspond to DNA fragments that were not cleaved by the T7
endonuclease. The 400 base pair band would correspond to the approximately similar sized DNA
fragments resulting from cleavage by the T7 endonuclease. The gel image in Figure 10, however,
did not produce the expected band sizes and was, in fact, an unsuccessful attempt.
More of the gRNA needed to be cloned in order to run the transfection, DNA isolation,
PCR, and T7 assays again, so the gBlock plasmid was used to transform OneShot cells which
would then replicate the plasmid. The replicated plasmid was then used to transfect mouse
embryonic stem cells a second time using the same protocol and the same cell splitting method.
PCR was run a second time on the genomic DNA isolated from the transfected cells and the
products were run on a gel shown in Figure 11. The gel image did not indicate that the expected
band of 800 base pairs was evident and so the T7 assay was not run a second time.
Though the T7 assay is a means to test if cells were cleaved by the Cas9 protein, it is not
the only, nor is it the most accurate test to determine if editing took place. The cells that were re-
plated after the second transfection were used to isolate RNA which was then used to make
cDNA for use in qPCR as shown in Figure 12. GAPDH was used as a positive control to
determine if there was a successful reduction in the expression of CTNNB1 (Beta-catenin) in
mouse embryonic stem cells. The mathematical calculations of the reduction in gene expression
are shown in the table below.
Sample Ct Beta-Catenin
(CTNNB1)
Ct GAPDH dCT ddCT 2^(-ddCT)
Untreated (Wild
Type) Cells
22.512 16.097 6.415 0 1
Edited Cells 23.692 16.390 7.302 0.887 0.541
The data from the qPCR assay suggests that the expression of CTNNB1 in mouse
embryonic stem cells was reduced by 45.9% through the process of genome editing via the
CRISPR-Cas9 complex. Though gene expression was not completely halted, the reduction in
expression indicates that there was some affect generated by the editing of the CTNNB1 gene at
the site chosen in exon 10. Exon 10 was chosen as the target for the Cas9 cleavage because it is

25. one of the first exons in the gene though it is downstream enough to hopefully be useful as a
gene expression reduction target.
The results produced by the editing of CTNNB1 in exon 10 generate several ideas for
future experiments. Selection of another exon for the target of Cas9 cleavage could potentially
reduce the expression of CTNNB1 further. Additionally, this experiment could be repeated with
the addition of allowing the mouse embryonic stem cells to develop further and monitoring the
changes in the development process. Furthermore, experiments could be, and certainly are being,
conducted to knockout expression of other genes to determine related pathways and resulting
affects. One potentially interesting future experiment could involve mutation of the HNH and
RuvC-like domains to eliminate their nickase abilities and allow the Cas9 protein to act as a
DNA binding protein alone. In doing so, the effects on replication and transcription that the Cas9
bound to DNA would produce could be cataloged.

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