Testing Guide Rnas to Activate the MAGP2 Promoter on Mouse Genomic DNA by CRISPR/Cas9

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:(all titles should be centered on the page above the respective section)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(citation?)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.(This is highly redundant- Can you reword it and still get the point across?)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 (awkward wording- If...then...).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. Ifthe expected results achieve from T7 assay,the laststep of this project would be testing the designed gRNA on mouse genomic DNA to activate the MAGP2 promoter.

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 plasmidswork onmouse genome.

Hypothesis:

Targeting MAGP2 promoteron 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.

Designing guide RNAs.Guide RNAs were designed by using CHOPCHOP. (

CRISPR gRNA Cloning. For insert prep(no abbreviations- should be preparation), 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 pX330-mM2G3 and pX330-mM2G12 were transformed into DH5α bacterial cells. Aftertransfection, 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, 30seconds, step 2. 95°C, 30seconds, step 3. 55°C, 30seconds, step 4. 68°C, 60seconds, 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.(type out your numbers under 100)

Results:

Five Guide RNAs were designed, but only two of them were tested in this project. The forward and reverse oligonucleotideswere 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.

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.

AB

Figure 2.CRISPR gRNA Cloning.A. The diagram shows different steps of 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.

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(awkward wording here, use the If...then...model to help clarify sentence). 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.

You may want to consider keeping all the figures together and keeping the text together. It gets a little overwhelming when you see figures then text, figures then text.

A

B- C

Figure 3.Designed gRNAs are tested to see if they cantargetthe 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 (these sentences are very repetitive- see if you can combine them or eliminate one of them to effectively get your point across), which cause mutation in genomic DNA. The mutated genomic DNA was tested with T7 assay, and the T7 samples were run on gel, whichshowed 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 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(are not achieved), the experiment should be continued by redesigning the primers.

If theexpected results achievefrom 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.

keep your figures to your results section- and maybe reference the figures when discussing the results

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.

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.

Review Form

Department of Biological Sciences

SaddlebackCollege, Mission Viejo, CA92692

Author (s): Zahra Abbasi

Title: Testing Guide RNAs to Activate the MAGP2 Promotor on Mouse Genomic DNA by CRISPR/Cas9

Summary

Summarize the paper succinctly and dispassionately. Do not criticize here, just show that you understood the paper.

Microfibril-associated glycoprotein 2 (MAGP2) is a small protein secreted into the extracellular matrix. If the protein is over expressed, it can lead to elastic, collagen, and blood vessel formation, which in turn can develop into a cancerous tumor. It is a known factor in the majority of ovarian cancer deaths. The hypothesis is that targeting the MAGP2 promoter on the mouse genomic DNA can activate the promoter and cause endogenous MAGP2 production. A CRISPR/Cas system was utilized using guide RNA to direct the Cas9 to the target DNA sequence, then the endonuclease cleaves the DNA at the specific target sequence. Mouse cells were then transfected with the guide RNA plasmids and several assays were run to determine functionality of the heteroduplexes. Further testing is required for the completion of this project, such as testing the completed gRNA sequences on the mouse genomic sequence to determine actual activation potential. At this point, there has been a successful creation and completion of two guide RNA plasmids that effectively identify the target sequence desired to activate the MAGP2 promoter.

General Comments

Generally explain the paper’s strengths and weaknesses and whether they are serious, or important to our current state of knowledge.

The paper is very thorough and full of detailed explanations of the various processes and tests run throughout the duration of the experiment. There is a very clear understanding of the topic you are discussing. The figures are very helpful in supplementing the complexity of your topic and providing a visual that makes the topic a little easier to understand. There is a logical flow to the paper, but revision of the results and discussion sections may be required. They are very repetitive and may need to be distinguished a little more. I realize this may be a challenge for this project since you have not completed the fullest extent of the project at this point. There are a few points throughout the paper where the details become redundant and you may want to cut and combine in a few places. This paper is highly relevant to current research and medical conditions including the ever present cancer diagnosis. This project, taken to the fullest extent of testing in the mouse genomic DNA could have major implications for cancer prevention or treatment in the future.

Technical Criticism

Review technical issues, organization and clarity. Provide a table of typographical errors, grammatical errors, and minor textual problems. It's not the reviewer's job to copy Edit the paper, mark the manuscript.

Spelling was not an issue, however, there were a few awkward sentences that need to be addressed for proper grammar. It takes away the fluidity of the paper when the reader must go back and re-read the sentence because of grammar issues. Organization of the paper was logical and flowed nicely. Additionally, most of the changes indicated are stylistic in nature and require a few minor changes. Pay attention to proper formatting when constructing papers now and in the future. Changes were indicated in red on this paper.

This paper was a final versionX This paper was a rough draft

Recommendation

 This paper should be published as is

 This paper should be published with revision

 This paper should not be published

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