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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저자는 이해 충돌에 조브의 정책을 읽고 공개 충돌이 더이 없다.

CRISPR / Cas9 매개 게놈 편집 기술은 게놈 수정에 대한 간단 빠르고 저렴한 방법을 제공한다. 기능 증강 특성에 대한 monoallelic 증강 삭제를 생성하고 분석하기 위해 여기에 설명 된 방법은 F1 마우스 세포에서 SNP를 활용합니다. 이러한 유형의 접근법의 이점은 : 1) monoallelic 인핸서 삭제가 중요한 증강제, 즉 두 대립 유전자에서 치사 셀에 이르는 조절 유전자의 단백질 수준에서 큰 감소를 삭제하...

전사 조절 요소에 의한 비정상적인 유전자 발현이 질병을 초래할 수있다 이러한 요소 개발 1 변형시 유전자 발현 시공간 미세 조정이 중요하다. 게놈 넓은 협회 연구에 의해 확인 된 많은 질병 관련 지역 비 코딩 지역에 있고 전사 증강 3-4의 기능을 가지고 있습니다. 강화제를 파악하고 그들이 자주 몇 킬로베이스 거리가 조절하는 유전자의 위치와 조직 - 특이 적 방식으로 5-6으로 활성화 될 수 있으므로 복잡 조절하는 유전자로 일치. 증강 예측은 일반적으로 히스톤 수정 마크, 중재자-cohesin 단지를 기반으로 세포 유형 특정 전사의 결합은 7-10 요인 있습니다. 예측 증강의 유효성 검사는 가장 자주 인핸서는 리포터 유전자 11 ~ 12의 발현을 활성화하는 벡터 기반의 분석을 통해 이루어집니다. 이러한 데이터는 V를 제공추정 인핸서 서열의 규제 가능성에 대한 aluable 정보는하지만 자신의 내생 게놈 맥락에서 그 기능을 공개하거나 조절하는 유전자를 식별하지 않습니다. 게놈 편집 기능 상실 분석에 의해 자신의 내생 적 맥락에서 전사 조절 인자의 기능을 연구하는 강력한 도구 역할을한다. 게놈 편집, 즉 CRISPR / Cas9 게놈 편집 시스템의 최근의 진보는, 게놈 기능의 조사를 용이하게한다. CRISPR / Cas9 시스템은 많은 생물학적 시스템에 사용하기 쉽고 적용 할 수 있습니다. Cas9 단백질 가이드 RNA (gRNA) (13)에 의해 게놈의 특정 사이트를 대상으로합니다. SpCas9 / gRNA 단지는 protospacer 인접 모티브 (PAM) 시퀀스, NGG 14 ~ 15에 &#39;5이어야 목표 게놈 시퀀스의 게놈을 스캔합니다. 목표에 gRNA의 gRNA에 상보적인 20 뉴클레오티드 (NT) 시퀀스의 염기쌍는 doubl 결과 SpCas9 클레아 활동을 활성화전자 가닥 브레이크 (DSB)가 PAM 시퀀스의 상류 3 염기쌍. 특이도가 gRNA 종자 지역의 전체 염기 쌍을 통해 달성되면, 6-12은 PAM에 인접한 NT; 반대로, 종자의 보통 16-17을 허용하는 &#39;5 불일치. 가입 비 동종 말 (NHEJ)의 DNA 수리 또는 상동 감독 수리 (HDR) mechanisms.NHEJ의 DNA 수리가 종종 방해 할 수있는 대상 사이트에서 몇 bp의 삽입 / 삭제 (삽입과 삭제)을 작성 중 하나를 소개 DSB는 복구 할 수 있습니다 유전자의 오픈 리딩 프레임 (ORF). 관심 영역의 측면 게놈 두 gRNAs에서 큰 결실을 생성하기 위해, 18 ~ 19을 사용할 수있다. 이 접근법은 궤적 제어 영역 또는 종래 증진제 9,18,20-22보다 큰 수퍼 증강제로 클러스터 전사 인핸서의 연구에 유용하다. Monoallelic 삭제는 전사의 시스 -regulation 연구를위한 귀중한 모델입니다. 관찰 장증강의 monoallelic 삭제 후 성적 수준에서 전자는 두 대립 유전자의 전사가 세포 적합성에 영향을 미치는 잠재적 영향을 때 발생할 수있는 혼란 효과없이 유전자 조절에서 해당 증강의 역할에 상관 관계. 감소 된 발현을 평가하기 그러나, 야생형 대립 유전자에서 삭제를 구별하는 능력없이 어렵다. 또한, 두 개의 대립 유전자를 구별하는 능력없이 각 대립 유전자의 결실을 유전형 특히이 PCR에 의해 전체 야생형 영역을 증폭하기 어렵다되는 1 메가 23&gt; 10킬로바이트 큰 결실에 대한 도전이다. 뮤스 castaneus으로 건너 뮤스의 musculus (129)에 의해 생성 된 F1 ES 세포의 사용은 두 가지 대립 유전자가 대립 유전자 특이 PCR 18,24에 의해 구별 할 수 있습니다. 이러한 세포의 하이브리드 유전자는 대립 유전자 특정 삭제 검사 및 발현 분석을 용이하게한다. 평균적으로이 두 게놈 사이의 모든 125 BP는 SNP에게있다, 표현과 유전자형에 대한 프라이머 설계 유연성을 제공하는 분석한다. 하나의 SNP의 존재는 프라이머 융해 온도 (Tm)에 영향을 미치는 두 대립 (25)의 식별을 허용 실시간 정량 PCR (qPCR에) 증폭 특이성을 타겟팅 할 수있다. 또한, 프라이머의 3 &#39;말단 내의 불일치 크게 원하지 대립 타겟 (26)의 증폭을 방지 프라이머로부터 연장하는 DNA 폴리머 라 아제의 능력에 영향을 미친다. CRISPR / Cas9 게놈 편집 시스템 (도 1)를 사용하여보다 큰 1킬로바이트의 특정 대립 유전자 인핸서 삭제 이후 발현 분석 F1 ES 세포의 사용은 다음의 프로토콜에 설명한다. <img alt="그림 1" src="/files/ftp_upload/53552/53552fig1.jpg" /> 시스 -reg을 연구하는 CRISPR / Cas9를 사용하여 그림 1. 증강 삭제유전자 발현의 위험률. 뮤스의 musculus 129 뮤스 castaneus 사이의 교차에 의해 생성 (A) F1 ES 세포는 대립 유전자 특정 삭제를 허용하는 데 사용됩니다. (B)는 두 개의 RNA를 가이드 (gRNA)는 인핸서 영역의 큰 Cas9 매개 결실을 유도하기 위해 사용된다. (C) 프라이머 세트가 많은 모노 - 및 이중 대립 유전자 결실을 식별하는 데 사용된다. 오렌지 프라이머 내부 프라이머이며, 보라색 프라이머는 외부 프라이머 및 녹색 프라이머는 gRNA 측면 프라이머되어 있습니다. 유전자 발현의 (D)의 변화는 대립 유전자 특정 qPCR에를 사용하여 모니터링됩니다. RFU 상대 형광 단위를 의미한다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53552/53552fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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.

여기에 설명 된 프로토콜 CRISPR / Cas9 게놈 편집 (도 1)를 이용하여 생성 monoallelic 인핸서 삭제 세포에서 유전자 발현의 시스 -regulation 공부 F1 ES 세포를 사용한다. 유전자형 유전자 발현의 gRNA 및 대립 유전자 특이 적 프라이머를 디자인이 방법에서 중요한 요소이다. 각 대립 유전자 특이 프라이머 세트는 대립 유전자 특이성을 확인하는 qPCR에 의해 검증되?...

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).

생성 CRISPR은 / Cas9 중재 Monoallelic 삭제할 경우는 마우스 배아 줄기 세포의 증강 기능을 연구하는

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

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

13.9K 조회수.  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).

비디오: 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.

증강 기능 다중 CRISPR 기반 증강 간섭 셀 라인에서의 해 부

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

연구

유전학

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.

재조합 효소 매개 카세트 교환을 사용하여 마우스 배아 줄기 세포의 구조 - 기능 연구

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 ...

발생학

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.

크로마틴 도메인의 드 노보 형성을 연구하는 방법

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.

인간 다능성 줄기 세포에서 CRISPR-CAS9를 사용하여 정의된 게놈 수정 생성

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.

급성 골수성 백혈병 세포에서 CRISPR/Cas9 리보뉴클레오단백질에 의한 RUNX1 내성 소음기의 전사 적 역할 조사

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.

유전자 조작 마우스 모델 개발을 위한 배아 줄기 세포에서 CRISPR/Cas9 매개 고효율 유전자 표적화

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.

접합체의 CRISPR 전기천공에 의한 마우스의 효율적인 게놈 편집

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.

질병 모델에서 조혈 세포의 연구를 위한 렌티바이러스 CRISPR/Cas9 매개 게놈 편집

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

면역학 및 감염병학

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.

CRISPR / Cas9를 통해 포유 동물 세포주의 게놈에 삭제 세대

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

생물학

CRISPR/Cas9 시스템을 사용한 Centromere-associated protein-e 유전자 편집

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.

저자 스포트라이트: CENP-E Knockout HeLa 세포 확립 – Kinesin-7 CENP-E 생물학 및 그 억제제를 연구하기 위한 새로운 접근 방식

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.

CRISPR/Cas9 시스템을 사용한 Centromere-Associated Protein-E CENP-E-/- Knockout 세포주 생성

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