Name: in vivo crispr/cas9 screening to simultaneously evaluate gene function in mouse skin and oral cavity
Uploaded: 2020-11-02T13:00:00.000+00:00
Duration: 7 min 52 s
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

이 작품은 캐나다 보건 연구소 (CIHR 365252), Krembil 재단 및 온타리오 연구 우수 라운드 8 (RE08-065)의 프로젝트 보조금에 의해 지원되었습니다. 삼패스 쿠마르 로가나단은 캐나다 암 학회 펠로우십(BC-F-16#31919)의 수혜자입니다.

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저자는 공개 할 것이 없습니다.

CRISPR/Cas9 게놈 편집은 유전자 기능 및 세포 과정을 조사하기 위하여 시험관 내 및 생체 내 연구 결과에서 널리 이용되었습니다. 대부분의 생체 내 연구는 CRISPR/Cas9 유전자 편집 세포를 동물 모델(동종 이식 또는 xenograft)으로 이식합니다. 이것은 암 유전학 및 세포 기능을 공부하는 강력한 도구이지만, 여전히 기본 조직 미세 환경이 부족하고 상처 및 / 또는 면역 반응을 유도 할 수 있습니다.
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게놈 이후 시대에 암 연구를 위한 도전의 한은 인과 유전자 돌연변이를 위한 게놈 데이터의 광대 한 양을 채굴하고 치료적으로 표적으로 할 수 있는 유전자 네트워크에 있는 노드를 확인하는 것입니다. 생물 정보 학적 분석은 이러한 목표를 향해 대단히 도움이되었지만, 시험관 및 생체 내 모델에서 효율적인 확립은 생물학적 시스템과 질병 상태의 복잡성을 해독하고 약물 개발을 가능하게하는 전제 조건입니다. 기존의 형질전환 마우스 모델은 생체 암 유전학 연구를 위해 광범위하게 사용되었지만, 그들의 비용, 시간 및 노동 집약적 인 성격은 현대 유전체학에 의해 해명 된 수백 개의 putative cancer 유전자의 체계적인 분석을 크게 금지했습니다. 이 병목 현상을 극복하기 위하여는, 우리는 한 마우스의 피부와 구강에 있는 유전자의 수백의 기능 손실 돌연변이를 동시에 유도하고 연구하기 위하여 CRISPR/Cas9 (정규로 간격짧은 Palindromic 반복군집실) 유전자 편집 기술3와 자궁 주입 방법론1,2에서 이전에 확립된 초음파 유도를 결합했습니다.
여기에서 기술된 방법론은 배아일 E9.5에서 살아있는 마우스 배아의 양수 구멍으로 설계한 렌즈바이러스의 초음파 유도 주입을 이용합니다. CRISPR/Cas9 성분이 함유된 렌티바이러스 화물은 단일 층 표면 엑토더름을 트랜스듀싱하여 나중에 피부와 구강의 상피를 초래합니다. 피부는 외부 표피로 구성되며 지하 막과 진피가 뒤따릅니다. 표피는 지하 막과의 접촉을 유지하고 증식 및 줄기 세포 용량을 가진 내부, 기저 층으로 구성된 계층화 된 상형입니다. 기저층은 가시, 세분화 및 층 각막 층2,4와같은 위의 차별화된 층을 야기한다. 리니지 추적 연구는 생체 내 CRISPR /Cas9 방법으로이 직접 성인기 전반에 걸쳐 지속되는 기저 층 내에서 조직 거주자 줄기 세포를 유전적으로 조작하는 것으로 나타났습니다. 렌티바이러스는 클론 밀도에서 E9.5 표면 ectoderm을 트랜스듀싱하기 위해 적정화될 수 있으므로, 이 방법은 수천 개의 이산 유전자 녹아웃 클론을 수용하는 모자이크 마우스를 생성하는 데 사용될 수 있다. 차세대 시퀀싱은 그 클론 내에서 CRISPR/Cas9 매개 유전자 절제의 효과를 복합화 된 방식으로5로분석하는 데 사용될 수 있다.
우리는 최근에 인간 머리와 목 편평상피세포암 (HNSCC)5에있는 재발하는 돌연변이를 보여주는 484개의 유전자의 기능을 평가하기 위하여 이 방법을 이용했습니다. HNSCC는 40-50%의 높은 사망률을 가진 파괴적인 암이고 세계6번째로 일반적인 암입니다. HNSCC는 상부 기도 또는 구강의 점막 안대기에서 발생하고 담배 와 알코올 소비 또는 인간 유두종 바이러스 (HPV) 감염과 관련이 있습니다. 피부 편평상피 세포 암 (SCC)은 피부 종양이며 인간7에서두 번째로 흔한 암을 나타낸다. 분리SCC 및 HNSCC는 조직학적 및 분자학적매우 유사하며, TP53, PIK3CA, NOTCH1 및 HRAS8에서변경을 나타내는 경우의 높은 비율을 가진다. 고주파에서 돌연변이된 유전자의 소수만이 있는 동안, 저주파에서 돌연변이된 유전자의 수백이 있습니다 (&5%), 일반적으로 긴 꼬리 분포로 불리는 현상. 긴 꼬리 유전자의 대다수가 생물학적 또는 임상 적 검증이 부족하기 때문에, 우리는 p53, Pik3ca 또는 Hras에서 민감하게 돌연변이를 가진 종양 경향이 마우스에서 이러한 유전자의 기능 상실을 모델링하기 위해 생체 내 CRISPR 스크리닝 기술을 사용하고 p53, Pik3ca 또는 Hras와 협력하는 몇 가지 새로운 종양 억제 유전자를 확인하여 종양발달을유발합니다 5.
여기서, 우리는 멀티플렉스 sgRNA 렌티바이러스 CRISPR sgRNA 라이브러리를 생성하고 마우스 표면 ectoderm에서 CRISPR/Cas9 유전자 녹아웃 스크린을 수행하는 상세한 프로토콜을 설명합니다. 참고로, 이러한 방법론은 CRISPR 활성화(CRISPRa) 및 CRISPR 불활성화(CRISPRi)와 같은 다른 유전자 조작 기술을 통합하거나 유전자 기능을 연구하기 위해 마우스의 다른 장기 시스템을 표적으로 변형하도록 조정할 수 있다.

<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

유전자 변형 마우스 모델 (GEMM)은 유전자 기능을 평가하고, 인간 질병을 모델링하고, 치료 방법을 평가하기 위한 전임상 모델로 봉사하는 데 중요한 역할을해 왔습니다. 그러나, 그들의 시간, 노동 및 비용 집약적인 성격은 유전자 기능의 체계적인 분석을 위한 그들의 유틸리티를 제한합니다. 최근 게놈 편집 기술의 발전은 이러한 한계를 극복하고 멀티플렉스및 신속한 방식으로 특정 마우스 기관 내에서 직접 특정 유전자 변류의 빠른 생성을 허용합니다. 여기서, 우리는 CRISPR/Cas9 기반 방법(정기적으로 간격이 있는 짧은 Palindromic 반복군)을 기술하여 마우스의 피부와 구강의 상피 내에서 수천 개의 유전자 녹아웃 클론을 생성하고 종양 억제기 유전자에 대한 생체 내 CRISPR 화면에서 직접 수행하는 데 필요한 단계를 자세히 설명하는   프로토콜을 제공합니다. 이러한 접근법은 조직 항상성 동안 또는 다양한 질병 환경에서 유전자의 생물학적 기능을 연구하기 위해 CRISPR 활성화 또는 CRISPR 불활성화와 같은 다른 기관 또는 기타 CRISPR/Cas9 기술에 적용될 수 있다.

도 1A는 단일 12k 또는 92k 올리고 칩에서 비용 효율적인 방식으로 여러 사용자 정의 CRISPR 라이브러리를 멀티플렉싱하기 위한 올리고뉴클레오티드의 설계를 나타낸다. sgRNA(파란색 코드)를 선택하면 올리고뉴클레오티드는 제한 부위(주황색 BsmBI) 및 라이브러리 특정 PCR 프라이머 쌍(녹색 색 코딩)으로 설계되었습니다. 여러 라이브러리는 단일 올리고 칩에서 멀티플렉스를 위한 ...

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

여기서 우리는 면역 능력 마우스의 피부와 구강에 있는 몇몇 유전자의 기능을 동시에 평가하기 위하여 자궁 배아 렌즈바이러스 주사에서 초음파 유도를 사용하여 생체 내 CRISPR/Cas9 검열 방법론을 기술합니다.

마우스 피부와 구강의 유전자 기능을 동시에 평가하기 위한 Vivo CRISPR/Cas9 스크리닝

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

in-vivo-crisprcas9-screening-to-simultaneously-evaluate-gene-function

Universidad San Sebastian

Research

JoVE Journal

Genetics

6.3K 조회수.  Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital. 여기서 우리는 면역 능력 마우스의 피부와 구강에 있는 몇몇 유전자의 기능을 동시에 평가하기 위하여 자궁 배아 렌즈바이러스 주사에서 초음파 유도를 사용하여 생체 내 CRISPR/Cas9 검열 방법론을 기술합니다.

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

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

CRISPR을 유전자형하기 위해 형광 PCR-모세관 젤 전기 영동 기술을 사용 / 높은 처리량 형식으로 녹아웃 돌연변이를 Cas9 매개

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

연구

유전학

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

CRISPR/Cas9를 사용 하 여 유전자 돌연변이 Calreticulin Cytokine 종속 조 혈 모 세포에서의 종양 활동을 조사 하기 위해 편집

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

암 연구학

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

감수 분열 동안 인간 유전자 기능을 평가 하기 위해 마우스 Oocytes를 사용 하 여 나

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

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

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

포유류 세포에 있는 풀린 CRISPR 기지를 둔 유전 스크린

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

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

난 모세포의 미세 주입을 통한 유전자 조작 마우스의 생성

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

발생학

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 ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

게놈 규모 CRISPR/Cas9 유전 스크린을 이용한 세포 표면 수용체 식별

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR 중재 손실 함수 분석 소 뇌과 립 세포를 사용 하 여 Utero에서 Electroporation 기반 유전자 이동

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

생쥐에서 유전자 카세트 knock-in 및 Flox 대립 유전자를 생성하기위한 접합체 미세 주입

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

디자인, 포장, 높은 역가 CRISPR 레트로와 렌티 배달 정위 주입을 통해

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...