Name: true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents
Uploaded: 2016-06-02T14:00:00.000+00:00
Duration: 9 min 18 s
Description: Watch this Scientific Journal Video about TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents at JoVE.com

This work was supported by Adaptable and Seamless Technology Transfer Program through Target-driven R&#38;D, Japan Science and Technology Agency, the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan, and the JSPS KAKENHI Grant Numbers 24300342 and 15H04313.

1. Construction of a Heptamer-type sgRNA Library
<ol>
	<li>Select a subset of the 16,384 (47) 7-nt sequences unless the full-scale library is to be constructed. 
	NOTE: The sequences can be random and/or designed to target specific cellular RNAs. For the latter, find hairpin structures resembling the T-arm of tRNA in a target RNA with the aid of an appropriate computer program and/or visually, and select sequences complementary to 7-nt sequences immediately downstream of the hairpin structures4,10,17,18.</li>
	<li>Synthesize each of the selected heptamers as a fully 2&#8242;-O-methylated, 5&#8242;- and 3&#8242;-phosphorylated RNA with a DNA/RNA synthesizer. Use the phosphoramidite method on the controlled-pore glass (CPG) support, and leave the 5&#8242;-terminal 4,4&#8242;-dimethoxytrityl (DMT) protecting group on in the last cycle (the addition of the 5&#8242; phosphate) of the synthesis23. 
	NOTE: Custom synthetic RNA can be also obtained from oligonucleotide suppliers.</li>
	<li>After completion of the last synthesis cycle, transfer the CPG with the synthesized oligonucleotide from the column to a glass vial with a Teflon-liner screw cap, add 1 ml of 29% ammonium hydroxide, and incubate it at 55 &#176;C for 8 hr to cleave the oligonucleotide from the CPG and remove the protecting groups from the bases and phosphates.</li>
	<li>Evaporate ammonium hydroxide with a centrifugal evaporator at 40 &#176;C and re-dissolve the oligonucleotide in 0.2 ml of 0.1 M n-hexylammonium acetate.</li>
	<li>Purify the synthetic oligonucleotide through reverse-phase high-performance liquid chromatography using a polystyrene-divinylbenzene column with a buffer containing 10-50% acetonitrile/0.1 M n-hexylammonium acetate. Run the column at 2.0 ml/min for 25 min at 80 &#176;C.</li>
	<li>Add 0.25-0.5 ml of concentrated acetic acid to the fractionated sample (1-2 ml) to make a 20% acetic acid solution, and incubate this solution for 1 hr at room temperature to remove the DMT group.</li>
	<li>Dry the sample with a centrifugal evaporator at 40 &#176;C, re-dissolve it in 1 ml of 29% ammonium hydroxide, and incubate the solution for 15 min at room temperature to remove the side chain of the 5&#8242; phosphate.</li>
	<li>Reduce the volume (1 ml) of the sample to about 0.3 ml with a centrifugal evaporator at 40 &#176;C.</li>
	<li>Dialyze the entire concentrated sample against distilled water (500 ml) using a dialysis tube of molecular weight cut-off 1,000 at 4 &#176;C overnight.</li>
	<li>Transfer the dialyzed sample from the dialysis tube to a 1.5-ml tube with a pipette, dry it with a centrifugal evaporator at 40 &#176;C, and subsequently re-dissolve it in water-for-injection-grade water, the volume of which should be small enough to make a greater than 100 &#181;M solution.</li>
	<li>Measure an optical density of the RNA sample at 260 nm with a spectrophotometer and adjust its concentration to 100 &#181;M by adding water-for-injection-grade water. Store the RNA solution at below -20 &#176;C before use.</li>
</ol>
2. Screening of the sgRNA Library for Cell Viability
<ol>
	<li>Prepare 103 cells/99 &#956;l/well (e.g., HL60 and RPMI-8226) on a 96-well dish in RPMI-1640 media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Prepare wells containing the media only for background subtraction. To eliminate edge effects, add sterile water to empty wells surrounding the sample wells.</li>
	<li>Add 1 &#956;l of heptamer-type sgRNA (100 &#956;M) dissolved in water-for-injection-grade water to each well. Assay each sgRNA in triplicate. Incubate the plate at 37 &#176;C in a 5% CO2 humidified incubator for 3 days.</li>
	<li>Add an appropriate volume of a commercially available WST-8 solution to each well. Incubate the plate at 37 &#176;C in the same incubator for 1 to 4 hr.</li>
	<li>Measure absorbance at 450 nm with a microplate reader. 
	NOTE: In most cases, linearity between absorbance and viable cell number appears to be maintained unless the absorbance value exceeds 1.0.</li>
</ol>


3. Evaluation of Effective sgRNAs by Flow Cytometry

<ol>
	<li>Prepare 103 cells/99 &#181;l/well (e.g., HL60) on a 96-well dish in RPMI-1640 media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Prepare at least 10 wells for one sgRNA to be tested. To eliminate edge effects, add sterile water to empty wells surrounding the sample wells.</li>
	<li>Add 1 &#181;l of heptamer-type sgRNA (100 &#181;M) dissolved in water-for-injection-grade water to each well. Incubate the plate at 37 &#176;C in a 5% CO2 humidified incubator for 3 days.</li>
	<li>Collect the cells treated with the same sgRNA from at least 10 wells into a 5-ml tube with a pipette. Spin the tube for 5 min in a centrifuge at 300 x g, and discard the media.</li>
	<li>Add 2 ml of 1x phosphate buffered saline (PBS) to the tube, and re-suspend the cells. Spin the tube for 5 min in a centrifuge at 300 x g, and discard PBS. Add 150 &#181;l of 1x annexin V binding buffer to the tube, and re-suspend the cells.</li>
	<li>Add 2 &#181;l of phycoerythrin (PE)-conjugated annexin V solution and 1 &#181;l of 7-aminoactinomycin D (7AAD) solution to the tube, mix them well, and incubate the tube for 15 min at room temperature in the dark. Analyze the sample using a flow cytometer24.</li>
</ol>

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A., Takaku, H., Tamura, M., Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+vascular+endothelial+growth+factor+expression+by+TRUE+gene+silencing.">Inhibition of vascular endothelial growth factor expression by TRUE gene silencing.</a> Biochem. Biophys. Res. Commun. 379, 924-927 (2009).</li><li>Sano, T., Takahashi, M., Nozaki, T., Takahashi, Y., Tamura, M., Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+utility+of+heptamer-type+sgRNA+for+TRUE+gene+silencing.">Expanding the utility of heptamer-type sgRNA for TRUE gene silencing.</a> Biochem. Biophys. Res. Commun. 416, 427-432 (2011).</li><li>Iizuka, S., Oridate, N., Nashimoto, M., Fukuda, S., Tamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+inhibition+of+head+and+neck+squamous+cell+carcinoma+cells+by+sgRNA+targeting+the+cyclin+D1+mRNA+based+on+TRUE+gene+silencing.">Growth inhibition of head and neck squamous cell carcinoma cells by sgRNA targeting the cyclin D1 mRNA based on TRUE gene silencing.</a> PLoS One. 9 (114121), (2014).</li><li>Takahashi, 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=Elimination+of+specific+miRNAs+by+naked+14-nt+sgRNAs.">Elimination of specific miRNAs by naked 14-nt sgRNAs.</a> PLoS One. 7 (38496), (2012).</li><li>Takahashi, 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+naked+RNA+heptamer+targeting+the+human+Bcl-2+mRNA+induces+apoptosis+of+HL60+leukemia+cells.">A naked RNA heptamer targeting the human Bcl-2 mRNA induces apoptosis of HL60 leukemia cells.</a> Cancer Lett. 328, 362-368 (2013).</li><li>Watanabe, N., 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=Induction+of+apoptosis+of+leukemic+cells+by+TRUE+gene+silencing+using+small+guide+RNAs+targeting+the+WT1+mRNA.">Induction of apoptosis of leukemic cells by TRUE gene silencing using small guide RNAs targeting the WT1 mRNA.</a> Leukemia Res. 37, 580-585 (2013).</li><li>Elbarbary, R. 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=Modulation+of+gene+expression+by+human+cytosolic+tRNase+ZL+through+5&#8242;-half-tRNA.">Modulation of gene expression by human cytosolic tRNase ZL through 5&#8242;-half-tRNA.</a> PLoS One. 4, 5908 (2009).</li><li>Elbarbary, R. 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=Human+cytosolic+tRNase+ZL+can+downregulate+gene+expression+through+miRNA.">Human cytosolic tRNase ZL can downregulate gene expression through miRNA.</a> FEBS Lett. 583, 3241-3246 (2009).</li><li>Ninomiya, 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=Potential+small+guide+RNAs+for+tRNase+ZL+from+human+plasma,+peripheral+blood+mononuclear+cells,+and+cultured+cell+lines.">Potential small guide RNAs for tRNase ZL from human plasma, peripheral blood mononuclear cells, and cultured cell lines.</a> PLoS One. 10, 0118631 (2015).</li><li>Takahashi, 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=Screening+of+a+heptamer-type+sgRNA+library+for+potential+therapeutic+agents+against+hematological+malignancies.">Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies.</a> Leukemia Res. 38, 808-815 (2014).</li><li>Shivarov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRUE+gene+silencing+for+hematologic+malignancies.">TRUE gene silencing for hematologic malignancies.</a> Leukemia Res. 38, 729 (2014).</li></ol>

Niigata University of Pharmacy and Applied Life Sciences has a pending patent application on a subset of the heptamer-type sgRNAs.

In most cases, fully 2&#8242;-O-methylated, 5&#8242;- and 3&#8242;-phosphorylated heptamer-type sgRNAs dissolved in water-for-injection-grade water (in the concentration of 100 &#181;M) appear to be stable at below -20 &#176;C for at least one year, judging from their activity to induce apoptosis in cancer cells. However, their stability would change depending on their sequence, quality, and purity. In some cases, 5&#8242;- or 3&#8242;-phosphate of a part of sgRNA molecules appears to be removed spontaneously during storage, judging from mass spectrometry analysis, although this may not affect their activity seriously.
When a hit sgRNA is obtained by the library screening, its cellular target RNAs can be found by transcriptome analysis through a DNA microarray and/or next-generation sequencing. The data obtained so far, however, suggest that downregulation levels of target RNAs via heptamer-type sgRNAs are usually moderate regardless of the presence or absence of transfection reagents10,12,14,17,18. 
The advantage of heptamer-type sgRNAs targeting BCL2 and WT1 mRNAs is that they can efficiently induce apoptosis in human leukemia cells in spite of weak downregulation of the target mRNAs17,18, suggesting the presence of additional RNA targets and/or additional mechanisms other than TRUE gene silencing. 
Since even the screening of the library containing only 156 heptamer-type sgRNAs can make 20 hits, around 2,000 hits would be expected to be obtained by screening the full-scale library composed of 16,384 heptamers22,25. 
For future applications, sgRNAs other than the heptamer type can be more suitable for TRUE gene silencing11-13,15,16, although heptamer-type sgRNAs are easier, more accurate and cheaper to synthesize than longer sgRNAs. Once the therapeutic sgRNA candidate is obtained, it is easy to proceed to animal model experiments.

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies1. This technology has been developed based on properties of tRNase ZL (or 3&#8242; tRNase), tRNA 3&#8242; processing endoribonuclease: it can cleave any target RNA at any expected site under the direction of an artificial small guide RNA (sgRNA) by recognizing a pre-tRNA-like or micro-pre-tRNA-like structure formed between the target RNA and the sgRNA2-9. sgRNA, which is usually 7-31 nt in length, is categorized into four groups, 5&#8242;-half-tRNA, heptamer RNA, 14-nt linear RNA, and hook RNA.
We have demonstrated the efficacy of TRUE gene silencing by introducing various artificially-designed sgRNAs into living cells10-15. We have also shown that sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells16-18. TRUE gene silencing appears to work on the intracellular and intercellular gene regulatory network system via tRNase ZL and natural sgRNAs19-21.
We have constructed an sgRNA library containing 156 heptamer-type sgRNAs to search for potential therapeutic heptamer-type sgRNAs for blood cancers22. We have screened it for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. And 4 of them have been shown to reduce tumor growth rates in mouse xenograft experiments.
Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Analysis for sgRNA&#39;s cellular target RNAs and evaluation of effective sgRNAs in mouse xenograft models can be performed according to conventional methods17,22, although these are not included in this protocol.

<table><tbody><tr><td>Custom heptamer RNA</td><td>Nippon Bioservice</td><td></td><td></td></tr><tr><td>OligoSep Prep HC Cartridge</td><td>Transgenomic</td><td>99-3860</td><td></td></tr><tr><td>Water-for-injection-grade Water</td><td>Otsuka Pharmaceutical&amp;nbsp;</td><td>7131400A2129</td><td></td></tr><tr><td>RPMI-1640 medium</td><td>Wako</td><td>189-02025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>SIGMA-ALDRICH</td><td>172012-500ML</td><td></td></tr><tr><td>Penicillin-Streptomycin Mixed Solution</td><td>Nacalai Tesque</td><td>26253-84</td><td></td></tr><tr><td>96 Well Plate</td><td>Greiner Bio-one</td><td>655 180</td><td></td></tr><tr><td>Cell Counting Kit-8</td><td>DOJINDO</td><td>343-07623</td><td>WST-8 solution</td></tr><tr><td>Microplate Reader, Sunrise Thermo RC-R</td><td>TEKAN</td><td>510-82851</td><td></td></tr><tr><td>FACS Round-Bottom Tube</td><td>BD Falcon</td><td>60819-820</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>SIGMA-ALDRICH</td><td>P7059-1L</td><td></td></tr><tr><td>PE Annexin V Binding Buffer</td><td>BD Biosciences</td><td>51-66121E</td><td></td></tr><tr><td>PE Annexin V</td><td>BD Biosciences</td><td>51-65875X</td><td></td></tr><tr><td>7AAD</td><td>SIGMA-ALDRICH</td><td>A9400-1MG</td><td></td></tr><tr><td>Flow Cytometer, FACSCalibur</td><td>BD Biosciences</td><td>342973</td><td></td></tr></tbody></table>

true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

The six heptamer-type sgRNAs 5&#8242;-AUCUUCA-3&#8242; (H1885), 5&#8242;-ACACACA-3&#8242; (H3277), 5&#8242;-GGGGGCG-3&#8242; (H10927), 5&#8242;-GGGGCCC-3&#8242; (H10944), 5&#8242;-GCCCCCG-3&#8242; (H12287), and 5&#8242;-CACCAGC-3&#8242; (H13260) were chemically synthesized as 2&#8242;-O-methyl RNA containing 5&#8242;- and 3&#8242;-phosphates.
These sgRNAs were examined for the effects on viability of a human leukemia cell line, HL60, and a human myeloma cell line, RPMI-8226. Representative results of the cell viability assays are shown in Figure 1. The four sgRNAs H1885, H3277, H10927, and H13260 were very effective and reduced the viability of HL60 and RPMI-8226 cells by &#62;80% and &#62;70%, respectively. The heptamers H3277 and H10927 were also tested for the effects on non-cancerous HEK293 cell viability, and have been shown to hardly affect the cell viability22.
The flow cytometry with annexin V and 7AAD double staining was performed for HL60 cells treated with the heptamer H1885, H3277, H10927, or H13260. In each sgRNA, a total cell number (56-95%) in early and late apoptotic stages was much higher than that (4-6%) in mock (Figure 2). These observations suggest that the reduction of HL60 cell viability is due to apoptosis.
<img alt="Figure 1" src="/files/ftp_upload/53879/53879fig1.jpg" /> 
Figure 1. Cell viability assays. Cell viability was measured 72 hr after HL60 cells (A) or RPMI-8226 cells (B) were cultured in the absence or presence of 1 &#181;M of the naked heptamer-type sgRNA H1885, H3277, H10927, H10944, H12287, or H13260. The relative living cell numbers in the absence of sgRNAs are adjusted to 100. Error bars indicate SD (n = 3). *, P &#60; 0.002. Reprinted from Leukemia Research, Vol. 38, Takahashi M. et al., Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies, pp 808-815, Fig. 1, Copyright (2014), with permission from Elsevier. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53879/53879fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53879/53879fig2.jpg" /> 
Figure 2. Flow cytometry. With respect to HL60 cells, flow cytometry was performed with annexin V and 7AAD double staining. The cells were analyzed 72 hr after cultured in the absence or presence of 1 &#181;M of the naked heptamer-type sgRNA H1885 (A), H3277 (A), H13260 (A), or H10927 (B). Reprinted from Leukemia Research, Vol. 38, Takahashi M. et al., Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies, pp 808-815, Fig. 2, Copyright (2014), with permission from Elsevier. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53879/53879fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents at JoVE.com

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

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5.6K Views.  Niigata University of Pharmacy and Applied Life Sciences. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

Video: TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Cancer Research

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

Genetics

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Genome-Scale Screening to Identify and Compare Therapeutic Targets in Tumors

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

Author Spotlight: Finding New Therapeutic Targets for Malignant Peripheral Nerve Sheath Tumor Through Genome-Scale shRNA Screens 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

Biology

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

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Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells