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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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 ...

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