Name: crispr gene editing tool for microrna cluster network analysis
Uploaded: 2022-04-25T13:00:00
Description: Watch this Scientific Journal Video about CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis at JoVE.com

PC3-ML cell lines were kindly provided by Mark Stearn (Drexel University College of Medicine). Justin Toxey aided in PCR genotyping. This work was supported by a Breedan Adams Foundation Grant, a Ryan Translational Research Fund, and a Commonwealth Health Research Board Grant (CHRB-274-11-20) to AE-K.

1. Preparation for CRISPR gene editing and guide RNA design to generate miRNA cluster knockout cell lines
<ol>
	<li>Create a DNA file containing the complete genomic sequence of the miRNA cluster region of interest (intergenic, intronic) and at least 1 kB of surrounding genomic regions using a DNA sequence annotation software program19.
	<ol>
		<li>Use a feature editing tool in the created DNA file to mark the targeted miRNA cluster locus (intergenic, intronic) ...

<ol>
	<li>Hasegawa, T., Lewis, H., Esquela-Kerscher, A., Laurence, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Translating MicroRNAs to the Clinic. , 329-369 (2017).</li><li>Lewis, H., Esquela-Kerscher, A., Thiagalingam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Systems Biology of Cancer. , 134-153 (2015).</li><li>Esquela-Kerscher, A., Slack, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncomirs+-+microRNAs+with+a+role+in+cancer.">Oncomirs - microRNAs with a role in cancer.</a> Nature Reviews Cancer. 6 (4), 259-269 (2006).</li><li>Breving, K., Esquela-Kerscher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complexities+of+microRNA+regulation:+mirandering+around+the+rules.">The complexities of microRNA regulation: mirandering around the rules.</a> The International Journal of Biochemistry &amp; Cell Biology. 42 (8), 1316-1329 (2010).</li><li>Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+tools+for+microRNA+genomics.">miRBase: tools for microRNA genomics.</a> Nucleic Acids Research. 36, 154-158 (2008).</li><li>Kabekkodu, S. P., 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=Clustered+miRNAs+and+their+role+in+biological+functions+and+diseases.">Clustered miRNAs and their role in biological functions and diseases.</a> Biological Reviews. 93 (4), 1955-1986 (2018).</li><li>Xiang, J., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feud+or+friend?+The+role+of+the+miR-17-92+cluster+in+tumorigenesis.">Feud or friend? The role of the miR-17-92 cluster in tumorigenesis.</a> Current Genomics. 11 (2), 129-135 (2010).</li><li>Aqeilan, R. I., Calin, G. A., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-15a+and+miR-16-1+in+cancer:+discovery,+function+and+future+perspectives.">miR-15a and miR-16-1 in cancer: discovery, function and future perspectives.</a> Cell Death &amp; Differentiation. 17 (2), 215-220 (2010).</li><li>Hasegawa, T., 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=Characterization+and+evidence+of+the+miR-888+cluster+as+a+novel+cancer+network+in+prostate.">Characterization and evidence of the miR-888 cluster as a novel cancer network in prostate.</a> Molecular Cancer Research. , (2018).</li><li>Lewis, H., 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=miR-888+is+an+expressed+prostatic+secretions-derived+microRNA+that+promotes+prostate+cell+growth+and+migration.">miR-888 is an expressed prostatic secretions-derived microRNA that promotes prostate cell growth and migration.</a> Cell Cycle. 13 (2), 227-239 (2014).</li><li>Xu, J., 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=Evidence+for+a+prostate+cancer+susceptibility+locus+on+the+X+chromosome.">Evidence for a prostate cancer susceptibility locus on the X chromosome.</a> Nature Genetics. 20 (2), 175-179 (1998).</li><li>Schleutker, J., 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=A+genetic+epidemiological+study+of+hereditary+prostate+cancer+(HPC)+in+Finland:+frequent+HPCX+linkage+in+families+with+late-onset+disease.">A genetic epidemiological study of hereditary prostate cancer (HPC) in Finland: frequent HPCX linkage in families with late-onset disease.</a> Clinical Cancer Research. 6 (12), 4810-4815 (2000).</li><li>Farnham, J. M., Camp, N. J., Swensen, J., Tavtigian, S. V., Albright, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confirmation+of+the+HPCX+prostate+cancer+predisposition+locus+in+large+Utah+prostate+cancer+pedigrees.">Confirmation of the HPCX prostate cancer predisposition locus in large Utah prostate cancer pedigrees.</a> Human Genetics. 116 (3), 179-185 (2005).</li><li>Brown, W. 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=Hereditary+prostate+cancer+in+African+American+families:+linkage+analysis+using+markers+that+map+to+five+candidate+susceptibility+loci.">Hereditary prostate cancer in African American families: linkage analysis using markers that map to five candidate susceptibility loci.</a> British Journal Of Cancer. 90 (2), 510-514 (2004).</li><li>Bochum, 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=Confirmation+of+the+prostate+cancer+susceptibility+locus+HPCX+in+a+set+of+104+German+prostate+cancer+families.">Confirmation of the prostate cancer susceptibility locus HPCX in a set of 104 German prostate cancer families.</a> Prostate. 52 (1), 12-19 (2002).</li><li>Doudna, J. A., Charpentier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing.+The+new+frontier+of+genome+engineering+with+CRISPR-Cas9.">Genome editing. The new frontier of genome engineering with CRISPR-Cas9.</a> Science. 346 (6213), 1258096 (2014).</li><li>Adli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRISPR+tool+kit+for+genome+editing+and+beyond.">The CRISPR tool kit for genome editing and beyond.</a> Nature Communications. 9 (1), (2018).</li><li>Jinek, 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=A+programmable+dual-RNA+guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337 (6096), 816-821 (2012).</li><li>. SnapGene&#174; software (from Insightful Science; available at snapgene.com) Available from: <a target="_blank" href="https://www.snapgene.com">https://www.snapgene.com</a> (2022)</li><li>. Ensembl Release 104 genome browser (from EMBL-EBI) Available from: <a target="_blank" href="https://www.ensembl.org">https://www.ensembl.org</a> (2022)</li><li>Howe, K. L., 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=Ensembl+2021.">Ensembl 2021.</a> Nucleic Acids Research. 49, 884-891 (2021).</li><li>. miRBase: the microRNA database, Release 22.1 (from Griffiths-Jones laboratory) Available from: <a target="_blank" href="https://www.mirbase.org">https://www.mirbase.org</a> (2022)</li><li>. Dharmacon CRISPR Design Tool (from Dharmacon) Available from: <a target="_blank" href="https://www.horizondiscovery.com/en/ordering-and-calculation-tools/crispr-dna-region-designer">https://www.horizondiscovery.com/en/ordering-and-calculation-tools/crispr-dna-region-designer</a> (2022)</li><li>. Off-Spotter (Venetia Pliatsika &amp; Isidore Rigoutsos of the Jefferson Computational Medicine Center) Available from: <a target="_blank" href="https://cm.jefferson.edu/Off-Spotter/">https://cm.jefferson.edu/Off-Spotter/</a> (2022)</li><li>Bao, X. R., Pan, Y., Lee, C. M., Davis, T. H., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+experimental+and+computational+analyses+of+off-target+editing+by+programmable+nucleases.">Tools for experimental and computational analyses of off-target editing by programmable nucleases.</a> Nature Protocols. 16 (1), 10-26 (2021).</li><li>. Dharmacon DharmaFECT Transfection Reagent Cell Type Guide. Choose Dharmacon DharmaFECT 1, 2, 3, or 4 for optimal transfection of siRNA or miRNA reagents Available from: <a target="_blank" href="https://horizondiscovery.com/-/media-Files/Horizon/resources/Selection-guides/dharmafect-cell-type-guidelines.pdf">https://horizondiscovery.com/-/media-Files/Horizon/resources/Selection-guides/dharmafect-cell-type-guidelines.pdf</a> (2022)</li><li>Baffoe-Bonnie, A. B., 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=A+major+locus+for+hereditary+prostate+cancer+in+Finland:+localization+by+linkage+disequilibrium+of+a+haplotype+in+the+HPCX+region.">A major locus for hereditary prostate cancer in Finland: localization by linkage disequilibrium of a haplotype in the HPCX region.</a> Human Genetics. 117 (4), 307-316 (2005).</li><li>Zhang, R., Peng, Y., Wang, W., Su, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+an+X-linked+microRNA+cluster+in+primates.">Rapid evolution of an X-linked microRNA cluster in primates.</a> Genome Research. 17 (5), 612-617 (2007).</li><li>Ohnuki, Y., Marnell, M. M., Babcock, M. S., Lechner, J. F., Kaighn, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+analysis+of+human+prostatic+adenocarcinoma+cell+lines.">Chromosomal analysis of human prostatic adenocarcinoma cell lines.</a> Cancer Research. 40 (3), 524-534 (1980).</li><li>Beheshti, B., Karaskova, J., Park, P. C., Squire, J. A., Beatty, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+high+frequency+of+chromosomal+rearrangements+in+the+centromeric+regions+of+prostate+cancer+cell+lines+by+sequential+giemsa+banding+and+spectral+karyotyping.">Identification of a high frequency of chromosomal rearrangements in the centromeric regions of prostate cancer cell lines by sequential giemsa banding and spectral karyotyping.</a> Molecular Diagnosis. 5 (1), 23-32 (2000).</li></ol>

This CRISPR gene editing procedure allows the investigator to quickly generate an entire panel of cell lines carrying unique miRNA cluster deletion combinations. The transfection of synthetic guide RNAs composed of 5&#39; and 3&#39; genomic site-specific crRNAs annealed with synthetic tracrRNA (1:1 molar ratio) in this protocol avoids time-consuming plasmid vector subcloning and allows for a more flexible and high-throughput experimental design using a 24-well format. The generation of cell lines carrying a DOX-inducible...

Better research tools are needed to investigate the contribution of noncoding RNAs in human disease. MiRNA dysregulation is often observed in human disorders such as cancer when comparing the expression profiles of these small noncoding RNAs in the tissues and body fluids (e.g., blood, urine) of cancer patients versus noncancer, healthy individuals, employing microarrays, quantitative real-time PCR (qRT-PCR), and next-generation deep sequencing technologies1,2. Recent work has characterized a large subset of these miRNAs as tumor suppressor, oncogenic, and metastasis factors that control tumor formation, disease progression, and drug resistance. Experimental overexpression and/or downregulation/loss of miRNAs result in functional and pleiotropic consequences in the cell, reflecting the wide range of cancer-associated activities these noncoding RNAs coordinate - growth, apoptosis, differentiation, chromatin remodeling, angiogenesis, epithelial to mesenchymal (EMT) and MET transitions, metabolism, and immune response3.
MiRNAs are encoded as single genes or reside in genomic clusters, which are transcribed in the nucleus and extensively processed before generating the biologically mature, single-stranded ~22 nucleotide (nt) miRNA species localized in the cytoplasm4. These small RNAs exert their effects post-transcriptionally and act as negative gene regulators that bind to messenger RNA (mRNA) targets in a sequence-specific manner to bring the catalytic RNA-induced silencing complex (RISC) to the mRNA site, resulting in mRNA degradation and/or a block in protein translation. MiRNAs are an extremely abundant class of noncoding RNAs in animal systems, and 2,654 mature miRNAs exist in the human genome (miRBase release 22.1)5. MiRNAs typically associate with incomplete complementarity to their mRNA targets4. Therefore, a single miRNA can regulate tens to hundreds of distinct mRNA targets and functionally impact a large range of biological pathways. To add to the complexity of miRNA-based mechanisms, a single mRNA can be regulated by multiple, distinct miRNAs. It is, thus, challenging to investigate how miRNA dysregulation disrupts the body&#39;s homeostatic balance and leads to human malignancy.
The majority of published studies have focused on a single miRNA when characterizing their role in disease events. However, ~30%&#160;of human miRNA genes are organized in clustered units (typically ~10 kilobases [kb]) that are often transcribed in the same orientation and co-expressed, indicating a coordinated and complex system of noncoding RNA regulation6. The largest polycistronic human miRNA cluster is the 14q32 cluster comprising 54 miRNA precursors. One of the most well-studied clustered miRNA units associated with human cancers is the miR-17-92 polycistronic cluster comprised of miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92-1 residing within intron 3 of the noncoding RNA, c13orf25. The miR-17-92 cluster is frequently amplified in hematopoietic malignancies and overexpressed in solid tumors and has established oncogenic roles in promoting cell cycle progression, apoptosis, and angiogenesis7. In addition, the tumor suppressive miR-15a and miR-16-1 cluster located within the intron of the noncoding gene Leu2 is often deleted in leukemias and downregulated in a large range of cancers, functioning to block tumor growth by targeting the antiapoptotic gene BCL2 and additional cell cycle progression genes8. The miR-888 cluster is elevated in patients with high-grade prostate cancer and consists of seven miRNA genes (miR-892c, -890, -888, -892a, -892b, -891b, and -891a) located on human chromosome Xq27.39,10.
The miR-888 cluster maps within the HPCX1 locus (Hereditary Prostate Cancer, X-linked 1) spanning Xq27-2, which was identified by linkage analysis of hereditary prostate cancer family pedigrees11,12,13,14,15,29. Functional characterization of individual miR-888 clustered members using conventional miRNA misexpression tools - miRNA mimics and antisense inhibitors - indicated that these miRNAs play overlapping roles in regulating prostate tumor growth and invasion9,10. However, these experimental methods do not lend themselves easily to studying how multiple miR-888 clustered members act synergistically or antagonistically in a noncoding RNA network to control tissue homeostasis and cancer progression. This described streamlined protocol using high-throughput CRISPR gene editing technology is modified to molecularly dissect miRNA clusters associated with human cancers (e.g., miR-888 cluster) to bridge this knowledge gap.
Bacterial CRISPR and CRISPR-associated (cas) genes mediate adaptive immunity against bacteriophages16. The discovery of this ancient procaryotic surveillance system was quickly adapted as an efficient scientific tool to easily target any desired genomic locus and make DNA sequence alterations within a large range of animal systems and cell types both in vitro and in vivo16,17. This technique holds great promise as an effective method to interrogate miRNA networks in the context of human disease. To this end, this high-throughput CRISPR gene editing protocol to study the miR-888 cluster spanning ~35 kb on human chromosome X in immortalized human prostate cancer cell lines (LNCaP, PC3-ML) is constructed to interrogate how cluster members coordinate cancer progression pathways. This approach can be applied to characterizing any miRNA cluster and allows investigators to rapidly generate human cell lines carrying the entire miRNA cluster deletion and individual and combination miRNA gene cluster deletions within a single experiment.
In this procedure, stable cell lines are established carrying a doxycycline (DOX)-inducible lentiviral expression system that enables the investigator to control Streptococcus pyogenes CRISPR-associated endonuclease Cas9 (csn1) gene expression through the constitutive DOX-inducible promoter TRE3G. The Tet-On 3G bipartite system involves a constitutive human elongation factor 1 alpha (hEF1alpha) promoter to drive the transcription of both the Tet-On 3G gene and the blasticidin resistance gene (BlastR) as a bicistronic transcript. The Tet-On 3G transactivator protein only binds to the TRE3G promoter in the presence of DOX, resulting in robust Cas9 transcription. In the absence of DOX, there is no or very minimal basal Cas9 expression. Therefore, the investigator can induce high Cas9 protein production in cells grown in media supplemented with DOX during the CRISPR gene editing steps and control for rapid CAS9 protein clearance upon DOX withdrawal.
This protocol also describes the design of synthetic CRISPR RNA (crRNA) oligonucleotides targeting regions flanking the entire miRNA cluster, individual miRNA hairpin (premiRNA) regions, and/or subsets of miRNA genes within the cluster. Each designed crRNA contains a unique 5&#39;-terminal 20 nt guide sequence (complementary to the genomic sequence of interest to be targeted), followed by an invariant 22 nt S. pyogenes repeat sequence (5&#39;-XXXXXXXXXXXXXXXXXXXX-GUUUUAGAGCUAUGCUGUUUUG-3&#39;) that enables base-pairing with the universal trans-activating CRISPR RNA (tracrRNA) oligonucleotides18. Together, the annealed crRNA and tracrRNA (mixed 1:1 ratio) function as the guide RNA for this protocol (Figure 1A). In each experiment, two synthetic guide RNAs are transfected into DOX-induced cells to associate and escort the bacterial Cas9 protein to the genomic DNA sites (5&#39; and 3&#39;) flanking the miRNA cluster region targeted for removal (Figure 1B).
A protospacer adjacent motif (PAM) sequence (5&#39;-NGG-3&#39; for wild type S. pyogenes Cas9) must be present in the cell genome and located immediately adjacent to the 20 nt DNA sequence targeted by the guide RNA17. The PAM sequence serves as a binding signal and positions the catalytic region of the endonuclease Cas9 enzyme on the targeted genomic DNA site, subsequently leading to directed, double-stranded (ds) DNA cleavage located ~3 nt upstream of the PAM. The cell&#39;s DNA repair machinery repairs the cleaved DNA ends, which can result in perfect ligation, but often nonhomologous end joining (NHEJ) occurs, causing small insertions or deletions (indels) at the repair site. Since miRNAs are noncoding genes often located within intergenic and intronic regions, these indels carry a low risk of creating unwanted nonsense/missense mutations.
By employing synthetic RNA oligonucleotides (annealed crRNA and tracrRNA, 1:1 molar ratio) encoding for the guide RNA complex in these experiments, this gene knockout strategy avoids time-consuming DNA vector subcloning and allows for huge flexibility in transfecting unique guide RNA combinations to cells in a 24-well format. Preparation of crude cell lysates for PCR genotype screening also avoids expensive and time-consuming DNA purification methods, while allowing for streamlined single colony cell line generation and phenotypic analysis. Indeed, this high-throughput CRISPR gene editing protocol has been used successfully to transfect cultured prostate cancer cell lines (LNCaP, PC3-ML) with 32 unique guide RNA combinations in a single experiment and generate knockout lines carrying deletions for the entire ~35 kb miR-888 cluster region; smaller deletion combinations for miR-888 cluster members belonging to the miR-743 and miR-891a families; as well as deletions for individual miRNA members within the miR-888 cluster. Studies like these will provide a clearer understating of how clustered miRNAs function cooperatively to regulate tumor growth, aggressiveness, and drug resistance before translating miRNAs to the clinic as therapeutic and diagnostic tools.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL PCR tubes, flat cap</td>
			<td>Fisher</td>
			<td>14-230-225</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Seal-Rite</td>
			<td>1615-5500</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>Corning Costar</td>
			<td>&#160;09761146</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>5x Phusion HF Buffer</td>
			<td>Thermo Scientific</td>
			<td>F-518L</td>
			<td>PCR reagent, genotyping</td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Fisher</td>
			<td>FB012927</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Falcon</td>
			<td>08-772-2C</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>Anti-CRISPR-Cas9 antibody [7A9-3A3], Mouse monoclonal</td>
			<td>AbCam</td>
			<td>ab191468</td>
			<td>Western blot reagent; 160 kDa; dilution 1:200</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x)</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td>Tisuue culture reagent</td>
		</tr>
		<tr>
			<td>BLAST nucleotide search engine</td>
			<td>National Center for Biotechnology Information</td>
			<td>NA</td>
			<td>Freeware; website: blast.ncbi.nlm.nih.gov</td>
		</tr>
		<tr>
			<td>Blasticidin S, Hydrochloride, Streptomyces griseochromogenes</td>
			<td>Calbiochem</td>
			<td>CAS 589205</td>
			<td>CRISPR reagent; Chemical, Working stock = 1 mg/mL in water</td>
		</tr>
		<tr>
			<td>Countess 3 Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>A50298</td>
			<td>Equipment; Cell counter</td>
		</tr>
		<tr>
			<td>Cryogenic tubes</td>
			<td>Thermo Scientific</td>
			<td>50001012</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>Dharmacon CRISPR Design Tool</td>
			<td>Horizon Discovery Ltd.</td>
			<td>NA</td>
			<td>Freeware; website: horizondiscovery.com/en/ordering-and-calculation-tools/crispr-dna-region-designer</td>
		</tr>
		<tr>
			<td>DharmaFECT siRNA Transfection Reagent #2</td>
			<td>Dharmacon, Inc.</td>
			<td>T-2002-02</td>
			<td>Transfection reagent; LNCaP and PC3-ML cell lines</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher</td>
			<td>&#160;BP231100</td>
			<td>Tisuue culture reagent</td>
		</tr>
		<tr>
			<td>DMEM Medium with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td>
			<td>Corning</td>
			<td>MT10013CV</td>
			<td>Tisuue culture reagent; Media for PC3-ML cells</td>
		</tr>
		<tr>
			<td>Doxycycline Hydrochloride, Ready Made Solution</td>
			<td>Sigma-Aldrich</td>
			<td>D3072-1ML</td>
			<td>CRISPR reagent; Chemical, Working stock = 1 mg/mL stock in water</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td>Tisuue culture reagent</td>
		</tr>
		<tr>
			<td>Edit-R CRISPR-Cas9 Synthetic tracrRNA, 20 nmol, designed</td>
			<td>Dharmacon, Inc.</td>
			<td>U-002005-20</td>
			<td>CRISPR reagent; Universal tracrRNA oligonucleotides</td>
		</tr>
		<tr>
			<td>Edit-R Modified Synthetic crRNA, 
			desalted/deprotected, 2 nmol</td>
			<td>Dharmacon, Inc.</td>
			<td>crRNA-460XXX</td>
			<td>CRISPR reagent; Designed crRNA oligonucleotides</td>
		</tr>
		<tr>
			<td>EditR Inducible Lentiviral hEF1aBlastCas9 Nuclease Particles, 50 &#956;L, 107 TU/mL</td>
			<td>Dharmacon, Inc.</td>
			<td>VCAS11227</td>
			<td>CRISPR reagent; Lenti-iCas9; Doxycycline-inducible lentiviral Streptococcus pyogenes Cas9 vector system</td>
		</tr>
		<tr>
			<td>Ensembl genomic viewer</td>
			<td>Ensembl</td>
			<td>NA</td>
			<td>Freeware; website: [ensembl.org] Use: Genome browser to identify a nucleotide seuqnces containing the miRNA cluster and surrounding gene/regulatory sequences.</td>
		</tr>
		<tr>
			<td>GAPDH Antibody (FL-335), rabbit polyclonal</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc25778</td>
			<td>Western blot reagent; 37 kDa; dilution 1:500</td>
		</tr>
		<tr>
			<td>Gel/PCR DNA Fragment Extraction Kit</td>
			<td>IBI Scientific</td>
			<td>IB47010</td>
			<td>Nucleic acid gel electrophoresis reagent</td>
		</tr>
		<tr>
			<td>Gibco Fetal Bovine Serum, certified</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>Tisuue culture reagent</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide</td>
			<td>MilliporeSigma</td>
			<td>H9268</td>
			<td>CRISPR reagent; Chemical, Working stock = 0.8 mg/mL</td>
		</tr>
		<tr>
			<td>Immobilon-FL PVDF Membrane</td>
			<td>MilliporeSigma</td>
			<td>IPFL10100</td>
			<td>Western blot reagent; nitrocellulose</td>
		</tr>
		<tr>
			<td>Intercept (TBS) Blocking Buffer</td>
			<td>LI-COR</td>
			<td>927-60001</td>
			<td>Western blot reagent</td>
		</tr>
		<tr>
			<td>IRDye 680RD Goat anti-Mouse IgG Secondary Antibody</td>
			<td>LI-COR</td>
			<td>926-68070</td>
			<td>Western blot reagent</td>
		</tr>
		<tr>
			<td>IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody</td>
			<td>LI-COR</td>
			<td>926-32211</td>
			<td>Western blot reagent</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>&#160;5425 R</td>
			<td>Plasticware</td>
		</tr>
		<tr>
			<td>miRBase microRNA viewer</td>
			<td>miRBase</td>
			<td>NA</td>
			<td>Freeware; website: [mirbase.org] Use: Borowser lists all annotated miRNA hairpins, mature miRNA sequences and associated clustered miRNAs mapping within 10 kb.</td>
		</tr>
		<tr>
			<td>NuPAGE 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well</td>
			<td>Invitrogen</td>
			<td>NP0321BOX</td>
			<td>Western blot reagent</td>
		</tr>
		<tr>
			<td>Odyssey CLx Imaging System</td>
			<td>LI-COR</td>
			<td>CLx</td>
			<td>Equipment; Western blot imaging</td>
		</tr>
		<tr>
			<td>Opti-MEM Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985062</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Owl D2 Wide-Gel Electrophoresis System</td>
			<td>Owl</td>
			<td>D2-BP</td>
			<td>Equipment; Nucleic acid gel electrophoresis system</td>
		</tr>
		<tr>
			<td>PCR Primers, designed (single-stranded DNA oligonucleotides)</td>
			<td>Integrated DNA Technology</td>
			<td>NA</td>
			<td>PCR reagent, genotyping</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase (2 U/&#181;L)</td>
			<td>Thermo Scientific</td>
			<td>F530S</td>
			<td>PCR reagent, genotyping</td>
		</tr>
		<tr>
			<td>RIPA Lysis Buffer 10x</td>
			<td>MilliporeSigma</td>
			<td>20-188</td>
			<td>Western blot reagent</td>
		</tr>
		<tr>
			<td>Proteinase K Solution (20 mg/mL), RNA grade</td>
			<td>Invitrogen</td>
			<td>25530049</td>
			<td>PCR reagent, genotyping</td>
		</tr>
		<tr>
			<td>RNase A, DNase and protease-free (10 mg/mL)</td>
			<td>Thermo Scientific</td>
			<td>EN0531</td>
			<td>PCR reagent, genotyping</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>MT10041CV</td>
			<td>Tisuue culture reagent; Media for LNCaP cells</td>
		</tr>
		<tr>
			<td>SnapGene Viewer</td>
			<td>Snap Gene</td>
			<td>NA</td>
			<td>Freeware; website: [snapgene.com]&#160;Use: DNA sequence annotation software program to create a DNA file for gRNA design and which&#160; highlights the miRNA cluster locus (intergenic, intronic), each individual miRNA hairpin sequence belonging to the miRNA cluster, and other nearby coding and non-coding genes and/or regulatory features</td>
		</tr>
		<tr>
			<td>TrypLE Select Enzyme (1x), no phenol red</td>
			<td>Gibco</td>
			<td>12563029</td>
			<td>Tisuue culture reagent; Recombinant trypsin</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Invitrogen</td>
			<td>16500100</td>
			<td>Nucleic acid gel electrophoresis reagent</td>
		</tr>
		<tr>
			<td>Veriti 96-Well Fast Thermal Cycler</td>
			<td>ThermoFisher Scientific</td>
			<td>4375305</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>XCell SureLock Mini-Cell Electrophoresis System</td>
			<td>Invitrogen</td>
			<td>EI0001</td>
			<td>Equipment; Protein gel electrophoresis system</td>
		</tr>
	</tbody>
</table>

crispr gene editing tool for microrna cluster network analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This high-throughput CRISPR deletion protocol was successfully employed using transfection of Cas9-inducible LNCaP and PC3-ML human cancer cell lines with synthetic oligonucleotide guide RNAs targeting the miR-888 cluster, which were studied in the context of prostate cancer. The miR-888 cluster was initially identified in an expression profiling screen as being elevated in prostate cancer patients with high-grade disease compared to low-grade and noncancer patients9,1...

Watch this Scientific Journal Video about CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis at JoVE.com

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...

This protocol uses a high throughput CRISPR gene editing workflow to molecularly dissect micro RNA genes organized and clustered units. And to determine how these noncoding RNA networks coordinate cancer progression pathways. This CRISPR gene editing procedure allows the investigator to quickly generate an entire panel of cell lines carrying unique micro RNA cluster deletion combinations while avoiding time consuming DNA vector sub cloning.This method will provide a clearer understanding of how clustered micro RNAs function cooperatively to regulate tumor growth, aggressiveness, and drug resistance before translating micro RNAs into the clinic as therapeutic and diagnostic tools. Demonstrating the procedure will be Grace Yi, a research assistant in my laboratory. To begin, create a DNA file containing the complete genomic sequence of the micro RNA cluster region of interest in at least one kilobytes of surrounding genomic regions using a DNA sequence annotation software program.Next, design four unique CRISPR RNAs for each targeted micro RNA locus. Two designed CRISPR RNAs to target complimentary DNA sequences immediately five prime of the micro RNA hairpin cluster and two designed CRISPR RNAs to target complimentary DNA sequences immediately three prime of the micro RNA hairpin cluster. Use a feature editing tool in the created DNA file to mark the DNA target sequence for each designed CRISPR RNA to be synthesized.Designed and synthesized PCR primers flanking the targeted micro RNA cluster regions for genotyping CRISPR cell lines. Perform a doxycycline concentration curve on newly generated lenti inducible Cas9 cell lines to determine the optimal conditions for Cas9 protein induction. Plate five times 10 of the fourth cells per well of each six well plate and grow at 37 degree Celsius, 5%carbon dioxide, in the preferred medium for 24 hours.Dilute dox in the preferred medium with a final dox concentration curve ranging from 0 5100, 150, 250 to 500 nanograms per milli liter. Label the wells of each seated six well plate from one to six. Remove the medium and replace with the appropriate dox concentrations.Grow plates one to four until 24 hours, 48 hours, and 72 hours dox induction and 120 hours post dox withdrawal. Harvest the wells of the plate at each time point. Process the cell pellets and ripa buffer for lysate isolation.Perform western blot analysis with 40 micrograms of protein to measure Cas9 protein induction and determine optimal Cas9 concentration. On paper, map out the five prime and three prime CRISPR RNA pair combinations that will be transected into each well of a 24 well plate targeting the entire micro RNA cluster, various clustered micro RNA gene combinations, as well as individual micro RNA cluster members. Plate the lenti inducible cast nine cell line at five times 10 to the fourth cells per well of a 24 well plate in the preferred medium containing the optimized dox concentration.Grow the cells for 24 to 48 hours at 37 degrees Celsius, 5%carbon dioxide to induce Cas9 protein expression. Transfect the lenti inducible Cas9 cells with a prepared five prime and three prime guide RNAs. Label four 1.5 milliliter micro centrifuge tubes per targeted micro RNA locus.In each tube, mix one to one molar ratio of tracer RNA and unique CRISPR RNAs together to form the two micromolar guide RNA complex. Add 2.5 microliters of tracer RNA, 1.25 microliters of the five prime positioned CRISPR RNA, 1.25 microliters of the three prime positioned CRISPR RNA, and five microliters of 10 millimolar tris hcl pH 7.5 buffer to a 1.5 milliliter centrifuge tube. Micro centrifuge for 30 seconds at 16, 000 times G to mix.Incubate at room temperature for five minutes. To each tube at 40 microliters of reduced serum medium to the 10 microliter of guide RNA complex reaction. Mix gently by pipetting up and down.In a clean 1.5 milliliter centrifuge tube, mixed two microliters of the transfection reagent and 48 microliters of reduced serum medium gently by pipetting up and down. Incubate at room temperature for five minutes. To each tube containing the 50 microliters of guide RNA mix, add the 50 microliters of the diluted transfection reagent.Pipette the mixture slowly up and down once to mix. Incubate at room temperature for 20 minutes. Add 400 microliters of antibiotic free medium supplemented with doxycycline to each tube containing the 100 microliters of the guide RNA transfection mix.Pipette gently to mix. Remove the medium from the doxycycline induced lenti inducible Cas9 cells. Add 500 microliters of media doxycycline guide RNA transformation mix based on the 24 well plate experimental map.Incubate the transfected cells for 48 hours at 37 degrees Celsius in 5%carbon dioxide. Replace the medium with the fresh prepared medium without doxycycline. Allow the cells to recover for another 24 to 48 hours at 37 degrees Celsius and 5%carbon dioxide.Harvest the transfected cells and prepare for genotyping and single cell delusions. Separate the 150 microliters of resuspended cells into three parts. Freeze one third of the transfected cells in medium plus 10%DMSO in cryo vials for long term storage.Transfer one third of transfected cells into a clean 0.2 milliliter PCR tube for genotyping. Prepare the final one third of transfected cells for counting and dilution in a 96 well plate format to generate single cell colonies. Using a 12 channel multi pipetter and a sterile reagent reservoir, add 100 microliters of diluted cells per well to two rows of the plate for each dilution.Allow four to six weeks for the cells to grow to cofluency for single cell colony expansion. Collect approximately 10 to 15 single cell colonies for genotyping. Identify at least three independent knockout single cell colony lines for each targeted micro RNA locus.Retain wild type single cell lines as controls. Resuspend been the cell pellet in four microliters of five X DNA polymerase buffer, one microliter of proteinase K, one microliter of RNase A, and nuclease free water up to a total volume of 20 microliters. Lice the cells in the thermo cycler at 56 degrees Celsius for 30 minutes and 96 degrees Celsius for five minutes.Store the cell lysates at minus 20 degrees Celsius until ready for PCR genotyping. Perform a PCR genotyping reaction using the designed PCR primers that flank the guide RNA five prime and three prime guide RNA targeted sites. Run the PCR reaction in a thermocycler using the program mentioned in the text manuscript.Load the PCR products onto a 1%agarose gel for electrophoresis analysis. Extract DNA from the isolated PCR fragments of the predicted molecular size for the knockout genotypes. Prepare the samples for DNA sequencing.Validate that the CRISPR reaction was successful and perform DNA sequencing of the isolated PCR fragments to identify the Cas9 cleavage site and confirm micro RNA locus deletion. Unique CRISPR RNAs were designed that targeted the entire 35 kilo bases miR-888 cluster region. Smaller cluster combinations within the miR-743 family or the miR-891 family as well as micro RNA deletions.Gel electrophoresis analysis of the PCR reactions indicated that the predicted DNA fragment size representing the knockout genotype was amplified for each CRISPR transfection. Single cell colonies were genotyped by PCR in sequence verified. DNA sequencing of these isolated knockout PCR fragments confirmed that the transfected five prime and three prime guide RNAs directed Cas9 cleavage ligation approximately three nucleotide upstream of the PAM sites and validated genomic loss of the targeted micro RNA locus.WST-1 proliferation assays comparing miR-891a knockout and wild type cells confirm that overexpression of micro RNA mimic lentiviral vector induces prostate cell growth and therefore it was predicted that miR-891 a loss would show reciprocal effects. Besides careful guide RNA design it is important to quickly generate single cell colonies through each micro RNA knockout line. It is especially relevant if the micro RNA knockout cells have reduced growth of viability and would be easily outcompeted in mixed populations with wild type cells.Additional methods such as quantitative realtime PCR, northern blot, and RNA sequencing should be performed to confirm that the CRISPR knockout cell lines do not express mature micro RNA for the deleted locus.

Research

JoVE Journal

Biology

Microarray Analysis for Saccharomyces cerevisiae

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

TGF-&#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene ...

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis

The Oriental fruit fly, Bactrocera dorsalis, is a highly invasive and adaptive pest species that causes damage to citrus and over 150 other fruit crops ...