Testing Guide RNAs to Activate the MAGP2 Promoter on Mouse Genomic
DNA by CRISPR/Cas9
‡
‡
Zahra Abbasi and †Alison Miyamoto, Ph.D.
Howard Hughes Medical Institute Research Scholar, †Department of Biological Science,
California State University Fullerton, Fullerton, CA 92831
Abstract: Microfibril-associated glycoprotein 2 (MAGP2) is a small protein that is secreted
from cells into the extracellular matrix. MAGP2 functions include inducing elastic and collagen
fiber formation, inducing blood vessel formation, and regulating cell signaling. The
overexpression of MAGP2 is involved in diseases like the advanced stage of papillary serous
ovarian cancer, which is responsible for the majority of the ovarian cancer deaths. A previous
study from the Miyamoto laboratory showed that the overexpressed MAGP2 can bind to
microfibrils and can be regulated by enzymatic cleavage. We are now interested to see if the
endogenous MAGP2 can bind to microfibril and be regulated by enzymatic cleavage. Therefore,
as a first step to activate endogenous MAGP2 expression, a CRISPR-Cas9 system was tested to
target the MAGP2 promoter for endogenous expression of MAGP2 in mouse cells.
CRISPR/Cas9 is a new method of genome editing by using guide RNA, and a CRISPR
associated (Cas) Endonuclease complex. The guide RNA directs the Cas9 to the target DNA
sequence, and the Cas9 endonuclease cuts the double stranded DNA at the target sequence. In
this project, two guide RNAs, mM2G3 and mM2G12 were designed and cloned into pX330
plasmids. By transfecting the mouse cells with pX330-mM2G3, pX330-mM2G12 plasmids, the
gRNA and Cas9 genes on plasmids were expressed. If gRNA-Cas9 complex targeted and cut the
MAGP2 promoter on mouse genomic DNA, which caused mutation in genomic DNA. The
mutated genomic DNAs were heated and incubated to form heteroduplexes, and then were tested
by T7 enzyme that digests heteroduplexes. After running the T7 assay samples on a gel,
unexpected bands were seen on the gel. Comparing the bands on the gel showed that the T7
endonuclease cut normal genomic DNA. In summary, this project has produced two guide RNA
plasmids, the pX330-mM2G3 and pX330-mM3G12. If the expected results achieve from T7
assay, the last step of this project would be testing the designed gRNA on mouse genomic DNA
to activate the MAGP2 promoter.
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Introduction:
Microfibril-associated glycoprotein 2 (MAGP2) is a small protein that is secreted from cells
into the extracellular matrix (Gibson et al., 1996). MAGP2 functions include inducing elastic and
collagen fiber formation, inducing blood vessel formation, and regulating cell signaling. MAGP2
is involved in some type of diseases like the advanced stage of papillary serous ovarian cancer,
which is responsible for the majority of the ovarian cancer deaths (Boring et al., 2009). MAGP2
staining intensity was correlated with the angiogenesis suggesting that MAGP2 would be a
proangiogenic factor in ovarian cancer (Mok et al., 2009).
A previous study of MAGP2 in our laboratory showed that the overexpressed MAGP2 can
bind to microfibrils and can be regulated by enzymatic cleavage (Miyamoto et al., 2014). Now,
we are interested to see if endogenous MAGP2 can produce and bind to microfibril by activating
the MAGP2 promoter through CRISPR/Cas.
Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR/Cas) is a microbial
nuclease system that protects the cell against invading phages and plasmids. CRISPR/Cas is used
as a new method of genome editing by using RNA, and a CRISPR associated (Cas)
Endonuclease complex (Cong et al., Science 2013). Guide RNA (gRNA) is a non-coding RNA
that can direct the Cas9 to the target DNA sequence, and Cas9 is an endonuclease that cut the
double stranded DNA at the target sequence. In this project, the designed guide RNAs were
tested in CRISPR/Cas9 system to see if the pX330-mM2G3, pX330-mM2G12 plasmids work on
mouse genome.
Hypothesis:
Targeting MAGP2 promoter on mouse genomic DNA by CRISPR/Cas9 can activate the
promoter and cause endogenous MAGP2 production.
Materials and Methods:
Cell line. T3 cell line derived from mouse ovarian tumor cells were used, which simulate the
human papillary serous ovarian carcinoma.
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Designing
guide
RNAs.
Guide
RNAs
were
designed
by
using
CHOPCHOP.
(https://chopchop.rc.fas.harvard.edu/)
CRISPR gRNA Cloning. For insert prep, the forward and reverse oligonucleotides were
annealed and phosphorylated, and for vector prep, pX330 was digested by BbsI restriction
enzyme. The prepared inserts were ligated to pX330 vectors. The resulting plasmids pX330mM2G3 and pX330-mM2G12 were transformed into DH5α bacterial cells. After transfection,
plasmids DNAs were purified by miniprep.
Amplifying mutated genomic DNA by PCR. The genomic DNA from T3 cells transfected with
pX330-mM2G3, pX330-mM2G12, and the genomic DNA from untransfected T3 cells were used
as templates, mM2P1F and mM2P2R as primers, and OneTaq enzyme as proofreading DNA
polymerase. GDNA PCR program was used to amplify the mutated genomic DNA with
following conditions: step 1. 95°C, 30 seconds, step 2. 95°C, 30 seconds, step 3. 55°C, 30
seconds, step 4. 68°C, 60 seconds, step 5. Repeat steps 2-4 a total of 30 cycles, step 6. 68°C, 5
minutes final extension.
T7 Assay. The PCR products were heated at 95°C for 5 minutes to denature the double stranded
DNAs and then incubated at room temperature for 60 minutes to anneal the single stranded
DNAs to form heteroduplexes. Each sample was divided into two tubes, and 1 ul diluted T7
enzyme (1U) was added to digested samples, and they incubated at 37°C for 20 minutes. After
incubation, samples were loaded on gel to check the quality of DNAs.
Results:
Five Guide RNAs were designed, but only two of them were tested in this project. The
forward and reverse oligonucleotides were annealed, phosphorylated and ligated to pX330
vectors that were digested with BbsI. Two plasmids pX330-mM2G3 and pX330-mM2G12 were
made, and transformed into DH5α bacterial cells. After transfection, plasmids DNAs were
purified by miniprep.
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Figure 1. Designed guide RNAs and primers. Five guide RNAs (red lines) and two primers
(blue lines) were designed. Two of the guide RNAs (bolded in the table) were tested in this
project. Each guide RNA can bind to the target DNA sequence on MAGP2 promoter (yellow
box) in mouse genome. The primers bind to the sequences that flank the target sequences were
guide RNAs bind.
A
B
Figure 2. CRISPR gRNA Cloning. A. The diagram shows different steps cloning to produce
multiple guide RNAs. B. Plasmid miniprep samples were run on the gel and the bands on the gel
showed the expected sizes with 260 bp.
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After transfecting T3 mouse cells by purified pX330-mM2G3 and pX330-mM2G12 plasmids,
the gRNA, and Cas9 genes were expressed. If Guide RNA directed the Cas9 to the target DNA
sequence, and the Cas9 cut the double stranded DNA at the target sequence. Both mutated, and
normal genomic DNAs were used as templates in genomic DNA PCR. The PCR products were
denatured and annealed to form heteroduplexes. Heteroduplexes are a double-stranded (duplex)
molecule of DNA with one normal strand and one mutated strand. The samples were divided into
digested and undigested, and T7 enzyme was added to digested samples in T7 assay. It was
expected that T7 endonuclease digest heteroduplexes. After incubation, both digested and
undigested samples were run on the gel. The expected size in undigested (-) lanes were 670 bp,
and the expected sizes in digested (+) lanes for T3+mM2G3: 308 bp and 355 bp, T3+mM2G12:
95 bp and 568 bp; and for T3 genomic DNA: 670 bp. The data showed unexpected bands on the
gel (Figure 3), and comparing digested and undigested samples on gel showed that the
unexpected bands were found in the normal genomic DNA lanes (Figure 3-C), which indicated
that T7 enzyme cut normal genomic DNAs.
A
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B- C
Figure 3. Designed gRNAs are tested to see if they can target the mouse genomic DNA. A.
The diagram shows all steps of testing designed gRNAs by transfecting mouse cells. B.
Amplified mutated genomic DNA showed the expected PCR products with 670 bp. C. T7
Endonuclease digest the heteroduplexes. Expected size in undigested (-) lanes: 670 bp. Expected
size in digested (+) lanes for T3+mM2G3: 308 bp & 355 bp; T3+mM2G12: 95 bp and 568 bp;
T3 genomic DNA: 670 bp.
Discussion:
pX330-mM2G3 and pX330-mM2G12 plasmids were made by ligating the mM2G3 and
mM2G12 inserts into digested pX330 plasmids. These plasmids can be used to transfect the
mouse cells and target the MAGP2 promoter on mouse genomic DNA to produce endogenous
MAGP2.
The mouse cells were transfected with pX330-mM2G3 and pX330-mM2G12 plasmids, which
cause mutation in genomic DNA. The mutated genomic DNA was tested with T7 assay, and the
T7 samples were run on gel, which showed unexpected bands on gel. Comparing the data
indicated that the bands were normal genomic DNAs that were cut instead of heteroduplexes by
T7 enzyme. Considering all steps of the experiment determined that the possible reasons for
these unexpected bands could be using the wrong amount of enzyme or buffer. According to the
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results, the next step of the experiment would be one of the following options: repeating the
experiment from amplifying the mutated genomic DNA with PCR, or repeating the experiment
from cell transfection; and if the expected results do not achieve, the experiment should be
continued by redesigning the primers.
If the expected results achieve from T7 assay, the last step of this project would be testing the
designed gRNA, mutant Cas9, and VPR complex on mouse genomic DNA to activate the
MAGP2 promoter.
Figure 4. Co-expressing the mutant Cas9 and guide RNAs in mouse cell. Guide RNAs and
mutant Cas9 will be expressed in mouse cell. The complex of gRNA, Cas9, and VPR binds to
target sequences on MAGP2 promoter in the mouse genome. It is expected that these complexes
activate the MAGP2 promoter.
Acknowledgment:
I would like to thank Dr. Alison Miyamoto for her support and guidance. I would also like to
thank the California State University Fullerton-Howard Hughes Medical Institute Research
Scholars Program.
References:
Chavez, Alejandro, Scheiman J., Vora, S., W Pruitt, B., et al. 2015. Highly efficient Cas9
mediated transcriptional programming. Nature America 12, 326 – 328.
Cong, Le, Ran, A., Cox, D., Lin, S., et al. 2013. Multiplex Genome Engineering Using
CRISPR/Cas Systems. Science 339, 819 – 823.
Mecham, Robert P., and M. A.Gibson. 2015 . The microfibril-associated glycoproteins (MAGPs)
and the microfibrillar niche. MATBIO- 1173, 21; 4C: 3, 6, 12, 13.
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Miyamoto, Alison, Donavan, L.J., Perez, E., Connett, B., Cervantes, R., Lai, K., Withers, G.,
Hogrebe, G. 2014. Binding of MAGP2 to microfibrils is regulated by proprotein convertase
cleavage. Matrix Biology 40, 27-33.
Mok, Samuel C., Bonome, T., Vathipadiekal, V., Bell, A., Johnson, M. E., et al. 2009. A Gene
Signature Predictive for Outcome in Advanced Ovarian Cancer Identifies a Survival Factor:
Microfibril-Associated Glycoprotein 2. Cancer Cell 16, 521-532.
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