We would like to thank all the members of the Mitchell lab for helpful discussions. This work was supported by the Canadian Institutes of Health Research, the Canada Foundation for Innovation and the Ontario Ministry of Research and Innovation (operating and infrastructure grants held by JAM).

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Die Autoren haben JoVE Politik über Interessenkonflikte und haben keine Konflikte lesen offen zu legen.

CRISPR / Cas9 vermittelte Genome Editing-Technologie bietet eine einfache, schnelle und kostengünstige Methode für die Genommodifikation. Die Methode, die hier detailliert zu erzeugen und monoallelic Enhancer Deletion für die funktionelle Charakterisierung Enhancer nimmt in F1 Mauszellen Vorteil von SNPs analysieren. Die Vorteile dieser Art von Ansatz sind: 1) monoallelic Enhancer Deletionen produzieren keine Verwechselung Wirkungen , die auftreten , wenn ein kritischer Enhancer aus beiden Allelen deletiert ist, ...

Transkriptions - Regulationselemente sind entscheidend für die räumlich-zeitliche Feinabstimmung der Genexpression während der Entwicklung 1 und Modifikation dieser Elemente in Krankheits 2 aufgrund aberrant Genexpression führen kann. Viele krankheitsassoziierten Regionen durch genomweite Assoziationsstudien identifiziert sind in nicht-kodierenden Regionen und verfügen über Funktionen von Transkriptionsverstärker 3-4. Identifizieren Enhancer und passender sie mit den Genen regulieren sie ist kompliziert , da sie oft mehrere Kilobasen entfernt von den Genen liegen sie regulieren und kann 5-6 in einer gewebespezifischen Weise aktiviert werden. Enhancer Prognosen werden auf Histonmodifikation Marken, Mediator-cohesin Komplexe häufig basiert und Bindung von zelltypspezifischen Transkriptionsfaktoren 7-10. Validierung des vorhergesagten Verstärker wird am häufigsten durch einen vektorbasierten Assay durchgeführt , in dem der Verstärker 11-12 Expression eines Reportergens aktiviert. Diese Daten liefern valuable Informationen über das regulatorische Potential mutmaßlicher Enhancer-Sequenzen, aber nicht offenbaren ihre Funktion in ihrer endogenen genomischen Kontext oder zu identifizieren, die Gene, die sie regulieren. Genome Bearbeitung dient als leistungsfähiges Werkzeug, um die Funktion von Transkriptionsregulationselemente in ihrer endogenen Zusammenhang mit loss-of-function-Analyse zu untersuchen. Jüngste Fortschritte in der Genombearbeitung, nämlich die CRISPR / Cas9 Genom Editing-System, ermöglichen die Untersuchung der Genomfunktion. Der CRISPR / Cas9 System ist einfach zu bedienen und anpassungsfähig für viele biologische Systeme. Das Cas9 Protein wird durch eine Führungs RNA (gRNA) 13 in das Genom an einer bestimmten Stelle ausgerichtet sind . Der SpCas9 / gRNA Komplex durchsucht das Genom für seine Ziel genomischen Sequenz , die 5 &#39;angrenzend Motiv (PAM) Sequenz, NGG 14-15 zu einem protospacer sein muss. Basenpaarung der gRNA zu seinem Ziel, eine 20 Nukleotid (nt) Sequenz, die komplementär zu der gRNA aktiviert SpCas9 Aktivität Nuklease in einem Doppe ergebe Strangbruch (DSB) 3 bp stromaufwärts der PAM-Sequenz. Die Spezifität wird durch vollständige Basenpaarung in der Seed-Region gRNA erreicht, die 6-12 nt neben dem PAM; Umgekehrt Mismatches 5 &#39;des Saatgutes werden üblicherweise 16-17 toleriert. Das eingeführte DSB repariert werden können, entweder durch die nicht-homologen Endverbindung (NHEJ) DNA-Reparatur oder Homologie gerichteten Reparatur (HDR) mechanisms.NHEJ DNA-Reparatur oft Einfügen / Löschen (indels) von wenigen bp an der Zielstelle erzeugt, die stören können der offene Leserahmen (ORF) eines Gens. Um größere Deletionen im Genom zwei gRNAs erzeugen, welche die Region von Interesse flankieren, können 18-19 verwendet werden. Dieser Ansatz ist besonders nützlich für die Untersuchung der transkriptionalen Enhancer geclustert in Locus - Kontrollregionen oder super-Enhancer , die größer sind als herkömmliche Enhancer 9,18,20-22. Monoallelic Streichungen sind ein wertvolles Modell für cis -Regelung der Transkription zu studieren. Die beobachtete change in Transkriptebene nach monoallelic Deletion eines Enhancers korreliert mit der Stellung dieser Enhancer in Genregulation ohne die verwirrende Wirkungen, die potenziell Zell fitness Beeinflussung beeinträchtigt wird, wenn die Transkription von beiden Allelen auftreten können. reduzierte Expression Auswertung ist schwierig, jedoch ohne die Fähigkeit, die gelöscht aus dem Wildtyp-Allel zu unterscheiden. Weiterhin ohne die Fähigkeit , an jedem Allel - Deletionen Genotypisierung die beiden Allele zu unterscheiden , ist eine Herausforderung, insbesondere für große Deletionen von&gt; 10 kb bis 1 Mb 23 , in dem es schwierig ist, die gesamte Wildtyp - Region durch PCR zu amplifizieren. Die Verwendung von F1 - ES - Zellen erzeugt durch Kreuzen Mus musculus 129 mit Mus castaneus können die zwei Allele durch allelspezifische PCR 18,24 unterscheiden. Das Hybrid-Genom in diesen Zellen erleichtert Allel-spezifische Deletion Screening und Expressionsanalyse. Im Durchschnitt gibt es eine SNP alle 125 bp zwischen diesen beiden GenomeFlexibilität für den Ausdruck und die Genotypisierung in Primer-Design, Untersuchungen. Die Anwesenheit eines SNP können die Primer Schmelztemperatur (T m) beeinflussen und Zielspezifität in real-time quantitative PCR (qPCR) Amplifikation unter Berücksichtigung Unterscheidung der beiden Allele 25. Weiterhin ist ein Mismatch innerhalb des Endes des Primers &#39;3 beeinflußt stark die Fähigkeit der DNA - Polymerase aus dem Primer verhindert Amplifikation des Allels unerwünschten Ziel 26 zu erstrecken. Beschrieben in dem folgenden Protokoll ist die Verwendung von F1 - ES - Zellen für die Allel - spezifische Enhancer - Deletionen von mehr als 1 kb und anschließende Expressionsanalyse unter Verwendung des CRISPR / Cas9 Genom Editiersystem (Abbildung 1). <img alt="Abbildung 1" src="/files/ftp_upload/53552/53552fig1.jpg" /> Abbildung 1. Enhancer Löschen mit CRISPR / Cas9 cis -REG zu studierenulation der Genexpression. (A) F1 ES durch eine Kreuzung zwischen Mus musculus sind 129 und Mus castaneus erzeugten Zellen für Allel - spezifische Löschen zu ermöglichen. (B) Zwei Führungs RNAs (gRNA) verwendet , um eine große Cas9-vermittelte Deletion des Enhancer - Region zu induzieren. (C) werden Primer - Sets verwendet , um große mono- und bi-allelische Deletionen zu identifizieren. Die orangefarbenen Primer sind die inneren Primer sind die lila Primer die äußeren Primer und die grünen Primer werden die gRNA flankierenden Primer. (D) Änderungen in der Genexpression verwenden allelspezifischen qPCR überwacht. RFU bezeichnet relative Fluoreszenzeinheiten. <a href="https://www.jove.com/files/ftp_upload/53552/53552fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www.jove.com/files/ftp_upload/53552/53552fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a>

<table><tbody><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>NEB</td><td>M0530S</td><td>high fidelity DNA polymerase used in gRNA assembly</td></tr><tr><td>Gibson Assembly Master Mix</td><td>NEB</td><td>E2611L</td><td></td></tr><tr><td>gRNA_Cloning Vector</td><td>Addgene</td><td>41824</td><td>A target sequence is cloned into this vector to create the gRNA plasmid</td></tr><tr><td>pCas9_GFP</td><td>Addgene</td><td>44719</td><td>Codon-optimized SpCas9 and EGFP co-expression plasmid</td></tr><tr><td>AflII</td><td>NEB</td><td>R0520S</td><td></td></tr><tr><td>EcoRI</td><td>NEB</td><td>R3101S</td><td></td></tr><tr><td>Neon Transfection System 100 &amp;micro;L Kit</td><td>Life Technologies</td><td>MPK10096</td><td>Microporator transfection technology</td></tr><tr><td>prepGEM</td><td>ZyGEM</td><td>PT10500</td><td>genomic DNA extraction reagent</td></tr><tr><td>Nucleo Spin Gel &amp;amp; PCR Clean-up</td><td>Macherey-Nagel</td><td>740609.5</td><td></td></tr><tr><td>High-Speed Plasmid Mini Kit</td><td>Geneaid</td><td>PD300</td><td></td></tr><tr><td>Maxi Plasmid Kit Endotoxin Free&amp;nbsp;</td><td>Geneaid</td><td>PME25</td><td></td></tr><tr><td>SYBR select mix for CFX</td><td>Life Technologies</td><td>4472942</td><td>qPCR reagent</td></tr><tr><td>iScript cDNA synthesis kit</td><td>Bio-rad</td><td>170-8891</td><td>Reverse transcription reagent</td></tr><tr><td>0.25% Trypsin with EDTA</td><td>Life Technologies</td><td>25200072</td><td></td></tr><tr><td>PBS without Ca/Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>0.5 M EDTA</td><td>Bioshop</td><td>EDT111.500</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies</td><td>14175095</td><td></td></tr><tr><td>1 M HEPES</td><td>Life Technologies</td><td>13630080</td><td></td></tr><tr><td>BSA fraction V (7.5%)</td><td>Life Technologies</td><td>15260037</td><td></td></tr><tr><td>Max Efficiency DH5&amp;alpha; competent cells</td><td>Invitrogen</td><td>18258012</td><td></td></tr><tr><td>FBS</td><td>ES cell qualified</td><td></td><td>FBS is subjected to a prior testing in mouse ES cells for pluripotency</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Glutamax</td><td>Invitrogen</td><td>35050</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>11960069</td><td></td></tr><tr><td>Pencillin/Streptomycin</td><td>Invitrogen</td><td>15140</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Invitrogen</td><td>11360</td><td></td></tr><tr><td>Non-essential aminoacid</td><td>Invitrogen</td><td>11140</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M7522</td><td></td></tr><tr><td>96-well plate</td><td>Sarstedt</td><td>83.3924</td><td></td></tr><tr><td>Sealing tape</td><td>Sarstedt</td><td>95.1994</td><td></td></tr><tr><td>CoolCell LX</td><td>Biocision</td><td>BCS-405</td><td>alcohol-free cell freezing container</td></tr><tr><td>CHIR99021</td><td>Biovision</td><td>1748-5</td><td>Inhibitor for F1 ES cell culture</td></tr><tr><td>PD0325901</td><td>Invivogen</td><td>inh-pd32</td><td>Inhibitor for F1 ES cell culture</td></tr><tr><td>LIF</td><td>Chemicon</td><td>ESG1107</td><td>Inhibitor for F1 ES cell culture</td></tr></tbody></table>

generating crispr/cas9 mediated monoallelic deletions to study enhancer function in mouse embryonic stem cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Das hier beschriebene Protokoll verwendet Zellen F1 ES cis -Regelung der Genexpression in monoallelic Enhancer deletiert Zellen zu untersuchen CRISPR / Cas9 Genom Bearbeitung (Figur 1) erzeugt. Die gRNA und Allel-spezifische Primer-Design für die Genotypisierung und der Genexpression sind die Schlüsselfaktoren in diesem Ansatz. Jedes Allel-spezifische Primer-Set muss von qPCR validiert werden Allel-Spezifität zu bestätigen. Allelspezifischen Primer , die nur...

Watch this Scientific Journal Video about Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells at JoVE.com

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Generieren von CRISPR / Cas9 Mediated monoallelic Löschungen Enhancer-Funktion in embryonalen Stammzellen der Maus zu studieren

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

generating-crisprcas9-mediated-monoallelic-deletions-to-study

Research

JoVE Journal

Developmental Biology

14.1K Aufrufe.  University of Toronto. Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Serie: Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these enhancers combine to produce a transcriptional response. As millions of enhancers have been identified, high-throughput tools are needed to determine enhancer function on a genome-wide scale. Current methods for studying enhancer function include making genetic deletions using nuclease-proficient Cas9, but it is difficult to study the combinatorial effects of multiple enhancers using this technique, as multiple successive clonal cell lines must be generated. Here, we present Enhancer-i, a CRISPR interference-based method that allows for functional interrogation of multiple enhancers simultaneously at their endogenous loci. Enhancer-i makes use of two repressive domains fused to nuclease-deficient Cas9, SID and KRAB, to achieve enhancer deactivation via histone deacetylation at targeted loci. This protocol utilizes transient transfection of guide RNAs to enable transient inactivation of targeted regions and is particularly effective at blocking inducible transcriptional responses to stimuli in tissue culture settings. Enhancer-i is highly specific both in its genomic targeting and its effects on global gene expression. Results obtained from this protocol help to understand whether an enhancer is contributing to gene expression, the magnitude of the contribution, and how the contribution is affected by other nearby enhancers.

This protocol describes the steps needed to design and perform multiplexed targeting of enhancers with the deactivating fusion protein SID4X-dCas9-KRAB, also known as enhancer interference (Enhancer-i). This protocol enables the identification of enhancers that regulate gene expression and facilitates the dissection of relationships between enhancers regulating a common target gene.

Dissektion der Enhancer-Funktion mit Multiplex CRISPR-basierte Enhancer Einmischung in Zell-Linien

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these ...

Forschung

Genetik

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Struktur-Funktions-Studien in embryonalen Maus-Stammzellen Rekombinase-vermittelten Kassettenaustausch

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Entwicklungsbiologie

A Method to Study de novo Formation of Chromatin Domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

A Method to Study de novo Formation of Chromatin Domains

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

Eine Methode zur Untersuchung der de novo Bildung von Chromatin-Domänen

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Generierung definierter genomischer Modifikationen mit CRISPR-CAS9 in humanen pluripotenten Stammzellen

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

Untersuchung der Transkriptionsrolle eines RUNX1 Intronic Schalldämpfers durch CRISPR/Cas9 Ribonucleoprotein in akuten myeloischen Leukämiezellen

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

CRISPR/Cas9-vermitteltes hocheffizientes Gen-Targeting in embryonalen Stammzellen zur Entwicklung genmanipulierter Mausmodelle

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.

Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.

Effiziente Genom-Editierung von Mäusen durch CRISPR-Elektroporation von Zygoten

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal ...

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Lentiviral CRISPR/Cas9-Vermittelte Genombearbeitung zur Untersuchung hämatopoetischer Zellen in Krankheitsmodellen

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Immunologie und Infektion

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

Generierung von Genomic Löschungen in Säugerzelllinien über CRISPR / Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Biologie

Zentromer-assoziierte Protein-E-Gen-Editierung mit dem CRISPR/Cas9-System

Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system.
Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors.
In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E&#160;knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

Autor im Rampenlicht: Etablierung von CENP-E-Knockout-HeLa-Zellen – Ein neuartiger Ansatz zur Untersuchung der Kinesin-7-CENP-E-Biologie und ihrer Inhibitoren

This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E&#160;knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.

Erzeugung von Zentromer-assoziierten Protein-E CENP-E-/- Knockout-Zelllinien mit dem CRISPR/Cas9-System

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing ...