Name: in vivo crispr/cas9 screening to simultaneously evaluate gene function in mouse skin and oral cavity
Uploaded: 2020-11-02T13:00:00
Description: 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

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>
	<li>Beronja, S., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-mediated+gene+function+analysis+in+skin.">RNAi-mediated gene function analysis in skin.</a> Methods in Molecular Biology. 961, 351-361 (2013).</li><li>Beronja, S., Livshits, G., Williams, S., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+functional+dissection+of+genetic+networks+via+tissue-specific+transduction+and+RNAi+in+mouse+embryos.">Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos.</a> Nature Medicine. 16, 821-827 (2010).</li><li>Shalem, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+CRISPR-Cas9+knockout+screening+in+human+cells.">Genome-scale CRISPR-Cas9 knockout screening in human cells.</a> Science. 343, 84-87 (2014).</li><li>Beronja, 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=RNAi+screens+in+mice+identify+physiological+regulators+of+oncogenic+growth.">RNAi screens in mice identify physiological regulators of oncogenic growth.</a> Nature. 501, 185-190 (2013).</li><li>Loganathan, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rare+driver+mutations+in+head+and+neck+squamous+cell+carcinomas+converge+on+NOTCH+signaling.">Rare driver mutations in head and neck squamous cell carcinomas converge on NOTCH signaling.</a> Science. 367, 1264-1269 (2020).</li><li>Leemans, C. R., Snijders, P. J. F., Brakenhoff, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+landscape+of+head+and+neck+cancer.">The molecular landscape of head and neck cancer.</a> Nature Reviews Cancer. 18, 269-282 (2018).</li><li>Green, A. C., Olsen, 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=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 ...

Research

JoVE Journal

Genetics

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...