This work was supported by a project grant from the Canadian Institute of Health Research (CIHR 365252), the Krembil Foundation and the Ontario Research Fund Research Excellence Round 8 (RE08-065). Sampath Kumar Loganathan is the recipient of a Canadian Cancer Society fellowship (BC-F-16#31919).

This protocol was approved and performed in accordance with IACUC of University of Toronto.
1. Design and cloning of pooled CRISPR libraries
<ol>
	<li>Select 4-5 sgRNAs targeting mouse genes of interest from resource such as the Broad Institute sgRNA designer (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) or CHOPCHOP server (https://chopchop.cbu.uib.no). Select an equal number of non-targeting sgRNAs from e.g., Sanjana et al.9 to generate ...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+squamous+cell+carcinoma:+an+epidemiological+review.">Cutaneous squamous cell carcinoma: an epidemiological review.</a> British Journal of Dermatology. 177, 373-381 (2017).</li><li>Campbell, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pathway+network,+and+immunologic+features+distinguishing+squamous+carcinomas.">pathway network, and immunologic features distinguishing squamous carcinomas.</a> Cell Reports. 23, 194-212 (2018).</li><li>Sanjana, N. E., Shalem, O., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+vectors+and+genome-wide+libraries+for+CRISPR+screening.">Improved vectors and genome-wide libraries for CRISPR screening.</a> Nature Methods. 11, 783-784 (2014).</li><li>Geraerts, M., Willems, S., Baekelandt, V., Debyser, Z., Gijsbers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+lentiviral+vector+titration+methods.">Comparison of lentiviral vector titration methods.</a> BMC Biotechnology. 6, 34 (2006).</li><li>Schramek, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+in+vivo+RNAi+screen+unveils+myosin+IIa+as+a+tumor+suppressor+of+squamous+cell+carcinomas.">Direct in vivo RNAi screen unveils myosin IIa as a tumor suppressor of squamous cell carcinomas.</a> Science. 343, 309-313 (2014).</li><li>Platt, R. 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=CRISPR-Cas9+knockin+mice+for+genome+editing+and+cancer+modeling.">CRISPR-Cas9 knockin mice for genome editing and cancer modeling.</a> Cell. 159, 440-455 (2014).</li><li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome Biology. 10, 25 (2009).</li><li>Li, W., 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=MAGeCK+enables+robust+identification+of+essential+genes+from+genome-scale+CRISPR/Cas9+knockout+screens.">MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens.</a> Genome Biology. 15, 554 (2014).</li><li>Winters, I. P., Murray, C. W., Winslow, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+quantitative+and+multiplexed+in+vivo+functional+cancer+genomics.">Towards quantitative and multiplexed in vivo functional cancer genomics.</a> Nature Reviews Genetics. 19, 741-755 (2018).</li><li>Rogers, Z. 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=Mapping+the+in+vivo+fitness+landscape+of+lung+adenocarcinoma+tumor+suppression+in+mice.">Mapping the in vivo fitness landscape of lung adenocarcinoma tumor suppression in mice.</a> Nature Genetics. 50, 483-486 (2018).</li><li>Chow, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV-mediated+direct+in+vivo+CRISPR+screen+identifies+functional+suppressors+in+glioblastoma.">AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma.</a> Nature Neuroscience. 20, 1329-1341 (2017).</li><li>Wang, G., 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=Mapping+a+functional+cancer+genome+atlas+of+tumor+suppressors+in+mouse+liver+using+AAV-CRISPR&#8211;mediated+direct+in+vivo+screening.">Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR&#8211;mediated direct in vivo screening.</a> Science Advances. 4, 5508 (2018).</li><li>Chan, K., Tong, A. H. Y., Brown, K. R., Mero, P., Moffat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pooled+CRISPR-based+genetic+screens+in+mammalian+cells.">Pooled CRISPR-based genetic screens in mammalian cells.</a> Journal of Visualized Experiments. (151), e59780 (2019).</li><li>Hart, 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=High-resolution+CRISPR+screens+reveal+fitness+genes+and+genotype-specific+cancer+liabilities.">High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities.</a> Cell. 163, 1515-1526 (2015).</li></ol>

CRISPR/Cas9 genome editing has been widely used in in vitro and in vivo studies to investigate gene functions and cellular processes. Most in vivo studies utilize CRISPR/Cas9 gene edited cells grafted into an animal model (allograft or xenograft). While this is a powerful tool to study cancer genetics and cellular functions, it still lacks the native tissue microenvironment and might elicit wounding and/or immune responses.
To overcome these challenges, several groups have pioneered direct in ...

One of the challenges for cancer research in the post-genomic era is to mine the vast amount of genome data for causal gene mutations and to identify nodes in the gene network that can be targeted therapeutically. While bioinformatic analyses have helped immensely towards these goals, establishing efficient in vitro and in vivo models is a prerequisite to decipher the complexity of biological systems and disease states and for enabling drug development. While conventional transgenic mouse models have been used extensively for in vivo cancer genetics studies, their cost-, time- and labor-intensive nature has largely prohibited the systematic analysis of the hundreds of putative cancer genes unraveled by modern genomics. To overcome this bottleneck, we combined a previously established ultrasound-guided in utero injection methodology1,2 with a CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing technology3 to simultaneously induce and study loss-of-function mutations of hundreds of genes in skin and oral cavity of a single mouse.
The methodology described here utilizes ultrasound-guided injections of engineered lentiviruses into the amniotic cavity of living mouse embryos at embryonic day E9.5. The lentiviral cargo containing CRISPR/Cas9 components transduce the single-layered surface ectoderm, which later gives rise to the epithelium of the skin and oral cavity. Skin is composed of an outer epidermis, followed by basement membrane and dermis. Epidermis is a stratified epithelium composed of an inner, basal layer, which maintains contact with the basement membrane and has proliferative and stem cell capacity. The basal layer gives rise to the differentiated layers above such as spinous, granular and stratum corneum layers2,4. Lineage tracing studies show that this direct in vivo CRISPR/Cas9 method genetically manipulates tissue-resident stem cells within the basal layer that persist throughout adulthood. As lentivirus can be titrated to transduce the E9.5 surface ectoderm at clonal density, this method can be used to generate mosaic mice harboring thousands of discrete gene knock-out clones. Next generation sequencing can then be used to analyze the effect of CRISPR/Cas9-mediated gene ablation within those clones in a multiplexed manner5.
We recently used this method to assess the function of 484 genes that show recurrent mutations in human Head and Neck Squamous Cell Carcinoma (HNSCC)5. HNSCC is a devastating cancer with a high mortality rate of 40&#8211;50% and it is the 6th most common cancer worldwide6. HNSCC arise in mucosal linings of the upper airway or oral cavity and are associated with tobacco and alcohol consumption or human papillomavirus (HPV) infection. Cutaneous Squamous Cell Carcinoma (SCC) are skin tumors and represent the second most common cancer in humans7. Cutaneous SCC and HNSCC are histologically and molecularly very similar, with a high percentage of cases exhibiting alteration in TP53, PIK3CA, NOTCH1, and HRAS8. While there are only a handful of genes mutated at high frequency, there are hundreds of genes found mutated at low frequency (&#60; 5%), a phenomenon commonly referred to as the long-tail distribution. As the majority of the long-tail genes lack biological or clinical validation, we used this in vivo CRISPR screening technology to model loss-of-function of these genes in tumor-prone mice with sensitizing mutations in p53, Pik3ca or Hras and identified several novel tumor suppressor genes that cooperate with p53, Pik3ca or Hras to trigger tumor development5.
Here, we describe a detailed protocol to generate multiplexed sgRNA lentiviral CRISPR sgRNA libraries and perform CRISPR/Cas9 gene knock-out screens in the mouse surface ectoderm. Of note, this methodology can be adapted to incorporate other gene manipulation technologies such as CRISPR activation (CRISPRa) and CRISPR inactivation (CRISPRi) or modified to target other organ systems in mouse to study gene functions.

<table>
	<tbody>
		<tr>
			<td>0.45 micron filter</td>
			<td>Sigma</td>
			<td>S2HVU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>12k or 92k oligo chip</td>
			<td>Customarray Inc. (Genscript)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm cell culture plates</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>293FT</td>
			<td>Invitrogen</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>293NT</td>
			<td>Systems Biosciences</td>
			<td>LV900A-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase</td>
			<td>NEB</td>
			<td>M0290L</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760-25</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>NEB</td>
			<td>9804S</td>
			<td></td>
		</tr>
		<tr>
			<td>BsmBI</td>
			<td>NEB</td>
			<td>R0580L</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromic gut suture</td>
			<td>Covidien</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deep sequencing (Next-Seq or Hi-Seq)</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA-cleanup kit</td>
			<td>Zymo Research</td>
			<td>D4008</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAesy Blood and Tissue DNA extraction kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		</tr>
		<tr>
			<td>Endura electrocompetent cells</td>
			<td>Lucigen</td>
			<td>60242-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>Drummond</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Speed Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>MLS-50</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Wisent Technologies</td>
			<td>800-011-LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P97</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-prep plasmid Kit</td>
			<td>Frogga Bio</td>
			<td>PDH300</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse oxygen anaesthesia system</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject II micromanipulator</td>
			<td>Drummond</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer 3.1 (Buffer for BsmBI)</td>
			<td>NEB</td>
			<td>R0580L</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle sharpener</td>
			<td>Sutter Instrument</td>
			<td>BV-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo cleanup kit</td>
			<td>Zymo research</td>
			<td>D4060</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE purified illumina sequencing primer</td>
			<td>IDT DNA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PEI (polyethyleneimine)</td>
			<td>Sigma</td>
			<td>408727-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Permoplast modeling clay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petridish with central opening</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 Polymerase 2x Master mix</td>
			<td>NEB</td>
			<td>M0494L</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Fluorometric Quantification</td>
			<td>Invitrogen</td>
			<td>Q33327</td>
			<td></td>
		</tr>
		<tr>
			<td>Semicircular Silicone plug</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone membrane</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo2000 ultrasound system</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo crispr/cas9 screening to simultaneously evaluate gene function in mouse skin and oral cavity

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct&#160;in vivo&#160;CRISPR&#160;screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.

Figure 1A shows the design of the oligonucleotides for multiplexing several custom CRISPR libraries in a cost-effective manner in a single 12k or 92k oligo chip. Once the sgRNAs (blue color coded) are selected, the oligonucleotides are designed with restriction sites (orange colored BsmBI) and library specific PCR primer pairs (green color coded). Several libraries can be designed by using unique combination of primer pairs for multiplexing in a single oligo chip. When PCR amplifying the lib...

Watch this Scientific Journal Video about In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity at JoVE.com

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Here we describe a rapid and direct in vivo CRISPR/Cas9 screening methodology using ultrasound-guided in utero embryonic lentiviral injections to simultaneously assess functions of several genes in the skin and oral cavity of immunocompetent mice.

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to ...

This protocol is significant because it allows researchers to simultaneously probe the tumor suppressive functions of hundreds of genes in head and neck cancer at a fraction of the cost of a conventional knockout mouse model. This technique is flexible and can be adapted to cover expression of genes to study their function in skin. The targeted CRISPR library protocol can also be applied for other cancer types.Place the anesthetized animal ventral side up on the heated surgery stage and use surgical tape to hold the paws in place. Apply the nosecone anesthesia tube for a constant supply of isoflurane and oxygen until the surgery is completed. To confirm E9.5 pregnancy, apply a small quantity of hair removal cream to the abdomen and to spread it over a three-by-three centimeter squared area using a cotton tipped applicator.After two to three minutes, gently remove the cream with gauze or tissue and wipe the area clean using PBS and 70%ethanol. Using the ultrasound probe and gel, check the pregnant mouse for embryonic day E9.5. This can also be done the day before at E8.5 to save time on the day of surgery.Remove the ultrasound gel and wipe the area clean using BPS and then 70%ethanol. Inject the most with subcutaneous analgesic. Then you sterile forceps and scissors to make a vertical incision in the skin.Use blunt forceps to separate the skin around the incision from the peritoneum. Then you sterile forceps and scissors to cut through the peritoneum. Using blunt forceps, gently pull out the left and right uterus horn and count the embryos.Reinsert most of the uterine horns, but leave the distal end of one uterine horn containing three embryos exposed. Using blunt forceps, gently push and pull the uterus with the three embryos, through the opening in the silicone membrane of the modified Petri dish. Stabilize the Petri dish using cubes of modeling clay.Stabilize the uterus with the three embryos inside the Petri dish, using a silicone mold. Then flatten the silicone plug at the side of the embryos using a blunt tool. Fill the Petri dish with sterile PBS and flush the silicone membrane on the bottom of the Petri dish, with the pregnant dam's belly, thereby preventing any leakage.Move the ultrasound head into the Petri dish, approximately 0.5 centimeters above the top embryo and adjust the stage so that the amniotic cavity becomes clearly visible in the ultrasound view. Align the injector needle, carefully into the modified Petri dish. Using the micromanipulator, place the tip of the needle within five millimeters of the top embryo, then toggle the needle back and forth to move into the plane where its tip appears the brightest in the ultrasound image.Using the micromanipulator, push the needle through the uterine wall into the amniotic cavity. Inject 62 nanoliters of the lentivirus with the sgRNA library at a slow injection speed. Press the Inject button eight times for a total of 496 nanoliters per embryo.Repeat the same procedure for the other two embryos. Then lift the ultrasound head and remove the silicone plug. Gently pushed the first two of the three embryos into the mouse abdomen using a sterile cotton swab.Pull out the next three embryos and push in the previously injected third embryo. Repeat the injection procedure until the desired number of embryos are injected, making sure to not exceed 30 minutes of anesthesia. When finished, lift the ultrasound head, remove the micromanipulator with the needle, aspirate the PBS and remove the Petri dish.Using sterile wipes, absorb any PBS that might have accumulated in the abdominal cavity. Close the peritoneal incision using absorbable sutures. Then use two staples to close the incision in the abdominal skin.Next, generation sequencing reads of PCR amplified DNA from plasmid and lentiviral library transduced cells should show a high correlation. If the sgRNA reads from plasmid DNA do not show equal representation of sgRNAs, then the viral preparation and concentration procedure must be repeated with care. The results of the ultrasound-guided injection of lentivirus carrying Cre recombinase in E9.5 Lox-STOP-Lox confetti mouse pups at postnatal day P0, are shown here.While high titer of virus would result in larger coverage of the mouse's skin, it would also result in transduction of multiple sgRNAs into the same cell. This plot shows next-generation sequencing reads of PCR-amplified DNA, from plasmid and lentiviral library transduced cells, as well as reads from four representative tumors. sgRNA guides targeting tumor suppressors ADAM10 and RIPK4 are enriched in the tumor samples compared to the plasmid pool or infected cells.Performing the surgery at E9.5 is critical. Remember to check for pregnancy with ultrasound, nine days after setting up the cross and identify the embryonic time point in each female mouse. Once the pups are born, the transduced cells can be isolated and extensively profiled using the latest gene profiling techniques such as single-cell RNA sequencing.

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6.2K visualizações.  Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital. This protocol is significant because it allows researchers to simultaneously probe the tumor suppressive functions of hundreds of genes in head and neck cancer at a fraction of the cost of a conventional knockout mouse model. This technique is flexible and can be adapted to cover expression of genes to study their function in skin. The targeted CRISPR library protocol can also be applied for other cancer types.Place the anesthetized animal ventral side up on the heated surgery stage an...