Name: using crispr/cas9 gene editing to investigate the oncogenic activity of mutant calreticulin in cytokine dependent hematopoietic cells
Uploaded: 2018-01-05T15:00:00
Description: Watch this Scientific Journal Video about Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells at JoVE.com

This work was supported by the NIH (R01HL131835), a Damon Runyon clinical investigator award and the Starr Cancer Consortium.

1. sgRNA Design Using Online Tools13
<ol>
	<li>Design sgRNAs targeting the gene of interest using freely available online tools.
	<ol>
		<li>Copy and paste the NCBI reference sequence of the gene of interest into the Broad Institute sgRNA designer web tool: (http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). 
		NOTE: This tool identifies sgRNA sequences with cleavage sites within exons and those that span the intron/exon boundary but still cleave wit...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+Gene-knockout+screens+and+target+identification+via+Whole+genome+sequencing+uncover+host+genes+required+for+picornavirus+infection.">CRISPR/Cas9-mediated Gene-knockout screens and target identification via Whole genome sequencing uncover host genes required for picornavirus infection.</a> J Biol Chem. 10, 1074 (2017).</li><li>Heckl, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mouse+models+of+myeloid+malignancies+with+combinatorial+genetic+lesions+using+CRISPR-Cas9+genome+editing.">Generation of mouse models of myeloid malignancies with combinatorial genetic lesions using CRISPR-Cas9 genome editing.</a> Nat Biotechnol. 32 (9), 941-946 (2014).</li><li>Daley, G. Q., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+an+interleukin+3-dependent+hematopoietic+cell+line+by+the+chronic+myelogenous+leukemia-specific+bcr/abl+protein.">Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific bcr/abl protein.</a> Proc Natl Acad Sci USA. 85, 9312-9316 (1988).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nat Protoc. 8 (11), 2281-2308 (2013).</li><li>Haeussler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+off-target+and+on-target+scoring+algorithms+and+integration+into+the+guide+RNA+selection+tool+CRISPOR.">Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.</a> Genome Biol. 17, 148 (2016).</li><li>Doench, J. 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(95), e52118 (2015).</li><li>Watanabe-Smith, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+acquired+mutations+in+transgenes+arising+in+Ba/F3+transformation+assays:+findings+and+recommendations.">Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays: findings and recommendations.</a> Oncotarget. 8, 12596-12606 (2017).</li><li>Fu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+CRISPR-Cas+nuclease+specificity+using+truncated+guide+RNAs.">Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.</a> NatBiotechnol. 32, 279-284 (2014).</li></ol>

Here we demonstrate the use of CRISPR/Cas9 gene editing to study the biological function of CALR mutations in hematopoietic cells. The success of this protocol is highly dependent on multiple factors. First, it is important to know what kinds of mutations may be responsible for the phenotype that is desired. In this protocol, the readout is the transformation of Ba/F3 cells to mIL-3 independence and the types of mutations are indels in exon 9 of CALR. However, if the desired mutation is a single base pa...

CRISPR/Cas9 technology has recently revolutionized targeted genome editing in living cells and organisms. It has become an extremely powerful tool for biomedical research and is currently being utilized as a potential avenue for therapy of genetic diseases1. The basis for all genome editing tools relies on the creation of a nuclease-induced DNA double stranded break (DSB) at the genomic locus to be modified. The DSBs can be repaired by non-homologous end-joining (NHEJ) or homology-directed repair (HDR)2,3. The advantage of the Cas9 nuclease over other genome engineering nucleases, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) is its dependence on RNA for targeting the nuclease to a desired DNA sequence, compared to the protein-DNA interactions found in ZFNs and TALENs2,3.
After the discovery of the CRISPR/Cas9 nuclease pathway as an adaptive immune system in prokaryotic cells4,5, much effort has gone into adapting the pathway for use in mammalian cell lines and model organisms2,3. As a tool for gene editing, the CRISPR/Cas9 pathway utilizes two main components: the Streptococcus pyogenes (Sp) derived Cas9 nuclease and sgRNAs targeting the gene of interest2,3. The sgRNA consists of 20 nucleotides that direct Cas9 to a specific site on the genome through RNA-DNA base pair complementarity2,3. The target site of the sgRNA must lie adjacent to a protospacer adjacent motif (PAM) site in the form of 5&#39; NGG, which is recognized by the SpCas9 nuclease. With these tools, Cas9 can be directed to any DNA sequence by designing sgRNAs that target the region of interest. In addition to Sp derived Cas9, there are additional variants for Cas9 with different features depending on the specific application. For example, there are Cas9 variants with higher specificity for on-target editing or single-strand cleavage capacity for DNA nicking6,7. Moreover, catalytically inactive Cas9 has recently been developed for transcriptional regulation8. Scientists have now used the CRISPR/Cas9 system for a variety of applications, such as gene knockin and knockout to study the biological functions of genes9, loss-of-function and gain-of-function library screens10 and genetic engineering of model organisms11.
In this protocol, we combine the CRISPR/Cas9 methodology with the Ba/F3 cellular transformation assay to understand the biological function of CALR mutations. Ba/F3 cells are a murine IL-3 dependent hematopoietic cell line that can be rendered IL-3 independent upon expression of certain oncogenes such as BCR-ABL12. In order to understand whether mutant calreticulin can transform Ba/F3 cells to cytokine independent growth, we targeted exon 9 of the endogenous Calr locus using CRISPR/Cas9 to introduce indel mutations and then withdrew IL-3 from the cells to apply a positive selection pressure, with the goal of recapitulating gain-of-function CALR mutations found in MPN patients. The protocol includes the design, cloning and delivery of sgRNAs, the development of stable Cas9 expressing cells and screening for CRISPR on-target gene editing. This protocol can be applied to different genes and various cytokine-dependent cell lines of interest and is especially valuable in modelling and studying the biological function of genes involved in cancer.

<table>
	<tbody>
		<tr>
			<td>BsmBI</td>
			<td>New England Biolabs</td>
			<td>R0580L</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Sigma Aldrich</td>
			<td>15025</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Life Technologies</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>Stbl3 cells</td>
			<td>Life Technologies</td>
			<td>C737303</td>
			<td></td>
		</tr>
		<tr>
			<td>TransIT-LT1</td>
			<td>Thermo Fisher Scientific</td>
			<td>MIR2300</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>51985034</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Thermo Fisher Scientific</td>
			<td>MT10040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Omega Scientific</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin/L-glutamine</td>
			<td>Life Technologies</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>mIL-3</td>
			<td>Peprotech</td>
			<td>213-13</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-VSV-G</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pLX_TRC311-Cas9</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene</td>
			<td>Sigma Aldrich</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>pXPR-011</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Genessee Scientific</td>
			<td>25-507</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>FERB49</td>
			<td></td>
		</tr>
		<tr>
			<td>LentiGuide-Puro</td>
			<td>Addgene</td>
			<td>Plasmid #52963</td>
			<td></td>
		</tr>
		<tr>
			<td>PNK</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
	</tbody>
</table>

using crispr/cas9 gene editing to investigate the oncogenic activity of mutant calreticulin in cytokine dependent hematopoietic cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Using the method outlined here, the goal of this experiment is to study the functional effects of introducing indel mutations to the endogenous Calr locus on hematopoietic cell transformation. The CRISPR/Cas9 system is used as a tool to create endogenous Calr mutations in Ba/F3 cells. Two sgRNAs were chosen to target exon 9 of Calr (Figure 1), in the region where insertions and/or deletion (indel) mutations typically occur in CA...

Watch this Scientific Journal Video about Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells at JoVE.com

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

The overall goal of this experiment is to utilize CRISPR/Cas9 gene editing to model calreticulin mutations in cytokine dependent hematopoietic cells in order to study their oncogenic activity. This method can help answer key questions in the hematological malignancies field such as the biological function of driver mutations when they are expressed at physiological levels. The main advantage of this technique is that it utilizes the CRISPR/Cas9 gene editing system to model and study the biological function of genes involved in cancer.To anneal and phosphorylate the oligos, add the reagents to a total of 10 microliters in a PCR tube kept on ice. Set the program in the thermocycler. Use water to dilute the reaction after cycle is complete.To digest the lentiGuide-Puro vector, prepare 50 microliters of digestor reaction. Then, start the digestion reaction at 55 degrees Celsius at a heat block. After one hour, add an additional microliter of BsmB1 restriction enzyme and continue the reaction for another hour.To dephosphorylate the cut back bone, add seven microliters of phosphatase reaction buffer and two microliters of phosphatase enzyme to the digestive vector. Then, incubate the mixture at 37 degrees Celsius for 30 minutes. Purify the already cut and dephosphylated back bone using a commercial kit.To prepare the ligation mixture, add 15 nanograms of digested back bone and other reagents to a PCR tube kept on ice and adjust the volume to 10 microliters with water. Incubate the reaction at room temperature for one hour. Then transform STBL3 compatible cells with two microliters of the ligation mixture.And then plate on the lysogeny broth agar plate containing 100 micrograms per milliliter of ampicillin. After this, incubate the plate overnight at 37 degrees Celsius. Pick three colonies and inoculate them.Perform mini prep for each culture and sequence the oligo insertion site with a U6 promoter region primer. Seed hek293t cells on a 10 centimeter dish. And leave it for overnight incubation.To prepare the DNA mixture for transfection, pipette 45 microliters of the transfection reagent to the DNA. Use a pipette to mix the DNA and transfection reagent carefully, and allow it to stand for 20 minutes. After this, add the DNA transfection mixture drop-wise on the seeded cells.Gently swirl the plates. And incubate at 5%carbon dioxide and 37 degrees Celsius for a day. Harvest the virus containing supernatent after 24 and 48 hours by passing it through a 0.22 micromiter filter.And aliquot 1.6 milliliters of supernatent in each cryo vial. Next aspirate the supernatent and resuspend the Ba/F3 cells in media by pipetting the cells up and down several times. Add 500 microliters of the resuspended cells, 1.5 milliliters of viral supernatant, four microliters of polybrene, and 10 nanograms per milliliter of murine interleukin 3 in a six well tissue culture plate.Centrifuge the plate at 440 G for 120 minutes at 37 degrees Celsius. After centrifugation, transfer the plate to an incubator for overnight incubation at 37 degrees Celsius with 5%carbon dioxide. After this, spin the cells down at 440 G for three minutes.And resuspend in five milliliters of fresh warm media in a six well plate. To select the Cas9 infected cells, first spin down the cell. And then resuspend in fresh warm media supplemented with five micrograms per milliliter of blasticidin after 48 hours of spinfection.Continue selecting cells for nine days with blasticidin til all the uninfected cells are dead. Then, transfer the resistant cells to a medium with one microgram per milliliter of blasticidin. Transduce the parental cells and the cells stably expressing Cas9 with pXPR-011 reporter construct through lentiviral infection.Continue selecting cells with two micrograms per milliliter of puromycin for three days, 48 hours after spinfection. Infect the Cas9 expressing cells with single guide ribonucleic acid using lentivirus. Then, use two micrograms per milliliter of puromycin to select the cells for three days, 48 hours post spinfection.Bin the exponentially growing Ba/F3 cells expressing ectopic Cas9 and the single guide ribonucleic acid of interest. Aspirate the supernatent and wash the cells with five milliliters of phosphate buffered saline. Spin the cells down, and wash with phosphate buffered saline three times.Then, count the number of cells using a cell viability analyzer. After this, seed cells in triplicates in two milliliters of fresh media without interleukin 3 in six well culture plates. Start monitoring and counting the cells every two days for eight days.Representative flow psychometric data demonstrating Cas9 activity in parental Ba/F3 cell and Ba/F3 overexpressing Cas9 cells is shown here. Green fluorescent protein signal is reduced by 76%in Ba/F3 overexpressing Cas9 cells, indicating robust Cas9 activity, whereas in parental Ba/F3 cells, the green fluorescent protein signal remains at 100%Representative growth curves for cells demonstrating oncogenic activity of calreticulin are shown here. CRISPR/Cas9 mediated plus one base pair frame shift mutation was introduced in the exon nine locus of calreticulin using m1 and m2 single guide RNase.Growth curves for calreticulin targeted Ba/F3 thrombopoietin receptor Cas9 cells shows murine interleukin 3 independent growth. However, the growth curves for the other cell types do not show any cytokine independent growth. Sequencing results confirm the on target editing of endogynous calreticulin exon nine in calreticulin targeted Ba/F3 thrombopoietin receptor Cas9 cells using the m1 and m2 single guide RNase.The plus one base pair frame shift mutations are indicated in black. Once mastered, this technique can be done in two weeks if performed properly. While attempting this procedure, it's important to understand what kind of mutations in the gene of interest may be responsible for the phenotype that is desired.Following this procedure, other methods to further characterize the mutation, such as its role in cell signaling or in protein protein interactions can be performed in order to further understand the mechanism by which is causes disease. After its development, this technique paved the way for researchers in the field of hematology to explore the effect of gain-of-function mutations at physiological levels on cell transformation in Ba/F3 hematopoietic cells. After watching this video, you should have a good understanding of how to utilize the CRISPR/Cas9 gene editing methodology to investigate the oncogenic activity of gain-of-function mutations in cytokine dependent hematopoietic cells.Don't forget that working with lentivirus can be extremely hazardous and precautions such as working in a BL2 plus facility should always be taken while performing this procedure.

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