저자는 모세관 전기 영동 실험을 도와 양 탄시 민, 미스 헬렌 옹, 박사 자오 이순신에게 감사의 말씀을 전합니다. 이 작품은 / 2,011분의 1,314 NMRC 및 환경부 AcRF 계층이 기금 보조금 MOE2011-T2-1-051을 부여 NMRC / IRG에 의해 지원되었다.

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+genome+engineering+with+programmable+nucleases.">A guide to genome engineering with programmable nucleases.</a> Nat. Rev. Genet. 15, 321-334 (2014).</li><li>Dahlem, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+methods+for+generating+and+detecting+locus-specific+mutations+induced+with+TALENs+in+the+zebrafish+genome.">Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome.</a> PLoS Genet. 8, e1002861 (2012).</li><li>Thomas, H. R., Percival, S. M., Yoder, B. K., Parant, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+genome+editing+and+phenotyping+facilitated+by+high+resolution+melting+curve+analysis.">High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis.</a> PLoS One. 9, 114632 (2014).</li><li>Kim, J. M., Kim, D., Kim, S., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+with+CRISPR-Cas-derived+RNA-guided+endonucleases.">Genotyping with CRISPR-Cas-derived RNA-guided endonucleases.</a> Nat. Commun. 5, 3157 (2014).</li></ol>

저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

선택의 모델 세포주에서 특정 유전자에서 노크는 유전자가 특정 세포 맥락에서하는 역할을 해명하기위한 일상이되었다. 사실, 여러 게놈 넓은 화면은 게놈 14, 15, 16에 거의 알려진 모든 인간의 유전자를 대상으로 CRISPR / Cas9 시스템을 사용하는 현재 사용할 수 있습니다. 이러한 대규모의 화면 (또는 관심의 개별 유전자의 대상?...

역방향 유전 방식은 과학자들이 셀 또는 전체 유기체의 게놈의 특정 변경의 효과를 규명 할 수있다. 예를 들어, 특정 유전자의 발현이 세포의 또는의 기능을 갖는다는 효과를 확인하기 위해 유전자 녹다운 (1, 2) (부분 감소) 또는 유전자 녹아웃 (3), 4 (완전 제거)에 의해 감쇠 될 수있다 유기체의 개발. 유전자 녹아웃 (knockout) 실험은 징크 핑거 뉴 클레아 제 (ZFNs) 및 전사 활성화 제와 같은 이펙터 클레아 (TALENs)와 같은 특정 프로그램 시퀀스 클레아의 도입 이후 쉽게되고있다. 그러나 정기적으로 짧은 회문 반복 (CRISPR)을 산재 클러스터 된의 상대적으로 최근의 특성화 / Cas9 시스템은 세대를 수행 할 수있는 전 세계 모든 실험실은 매우 간단했다전자 녹아웃 실험. 본질적으로, CRISPR / Cas9 시스템은 두 개의 필수 성분-A 인식 및 게놈의 특정 염기 서열에 상보성 단일 결합을 통해 가이드 RNA (sgRNA) 및 Cas9라고하는 효소로 구성된다. 게놈 DNA의 sgRNA-Cas9 복합체의 특이 적 결합 및 동작의 여파는 DNA의 이중 가닥을 절단한다. 이는 다시, 계속해서 비 - 상동 최종 접합 (NHEJ) 또는 동종 재조합 (HR) 경로를 통해 복구 된 세포에서 DNA 손상 반응 메커니즘을 트리거한다. NHEJ 수리 메커니즘 (그러나 HR 메커니즘이) 자주 삽입 / 삭제 (INDEL) 돌연변이의 결과로, 수리의 사이트에서 무작위로 삽입 또는 뉴클레오티드의 삭제를 초래하기 때문에, 엑손의 독서 프레임이 이동 될 수 있습니다. 이 다음에 의한 번역 및 넌센스 매개 붕괴 5, 6의 조기 종료에 유전자의 녹아웃 될 수 있습니다7. 유전자를 노크의 CRISPR / Cas9 시스템의 도입에 의해 제공되는 편리함에도 불구하고, 표적 세포의 클론의 유전자형은 특히 8, 9를 설정하는 높은 처리량, 병목 남아있다. 주요 본질적인 한계로 고통 또는 재정적 비용이 많이 드는 중 기존 기술. 예를 들어, 10 이중 가닥 DNA에서 불일치를 검출하는 효소 분석이다 검사원 또는 T7E1 분석은 야생형 클론 및 동형 접합성 돌연변이를 구별 할 수없는 이들 클론은 동일한 대립 유전자를 가지고 있고, 따라서 이후 (그 대립 동일 돌연변이 클론) 이들 DNA 서열 11 불일치를 표시하지 않는다. 또한, 높은 처리량 설정에서, 돌연변이 클론 유전형의 황금 표준으로 간주됩니다 생거 시퀀싱의 사용으로 인해 높은 비용 바람직하지 않다. 여기, 우리는 DET을 제시다른 유전자형 기존 기술의 한계를 회피하고 클레아 매개 녹아웃 클론의 높은 처리량 스크린을 수행하는데 특히 유용 할 수있는 형광 PCR 모세관 전기 영동 기술의 ailed 프로토콜. 이 방법을 수행 할 기술적으로 간단하고 시간과 비용을 절약 할 수 있습니다.

<table><tbody><tr><td>HEPG2 cells</td><td>ATCC</td><td>HB-8065</td><td></td></tr><tr><td>HyClone Dulbecco&#039;s Modified Eagles Medium (DMEM)</td><td>Thermo Fisher Scientific</td><td>SH30022.01</td><td></td></tr><tr><td>HyClone Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>SV30160.03</td><td></td></tr><tr><td>pSpCas9(BB)-2A-GFP plasmid</td><td>Addgene</td><td>PX458</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>11668027</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Thermo Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Trypsin-EDTA (0.5%), no phenol red</td><td>Thermo Fisher Scientific</td><td>15400054</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>HyClone Water, Molecular Biology Grade</td><td>GE Healthcare</td><td>SH30538.02</td><td></td></tr><tr><td>CRISPR sgRNA insert oligonucleotide (sense)</td><td>AITbiotech</td><td>None</td><td>Sequence: 5&#039;-CACCGCTAACCTTTCAGCCTGCCTA-3&#039;</td></tr><tr><td>CRISPR sgRNA insert oligonucleotide (anti-sense)</td><td>AITbiotech</td><td>None</td><td>Sequence: 5&#039;-AAACTAGGCAGGCTGAAAGGTTAGC-3&#039;</td></tr><tr><td>Unlabeled PCR amplification forward primer</td><td>AITbiotech</td><td>None</td><td>Sequence: 5&#039;-CACTAACTCCAATGCTTCAGTTTC-3&#039;; this primer is also used to sequence PCR amplified alleles</td></tr><tr><td>6-FAM-labeled fluorescent PCR forward primer</td><td>AITbiotech</td><td>None</td><td>Sequence: 5&#039;-6-FAM-CACTAACTCCAATGCTTCAGTTTC-3&#039;</td></tr><tr><td>HEX-labeled fluorescent PCR forward primer</td><td>AITbiotech</td><td>None</td><td>Sequence: 5&#039;-HEX-CACTAACTCCAATGCTTCAGTTTC-3&#039;</td></tr><tr><td>Unlabeled PCR reverse primer</td><td>AITbiotech</td><td>None</td><td>Sequence: 5&#039;-CCTCTTCCAAGTCTGCTTATGT-3&#039;</td></tr><tr><td>Taq PCR Core Kit</td><td>QIAGEN</td><td>201223</td><td></td></tr><tr><td>Hi-Di Formamide</td><td>Thermo Fisher Scientific</td><td>4311320</td><td></td></tr><tr><td>GeneScan 500 LIZ Dye Size Standard</td><td>Thermo Fisher Scientific</td><td>4322682</td><td></td></tr><tr><td>MicroAmp Optical 96-Well Reaction Plate</td><td>Thermo Fisher Scientific</td><td>4306737</td><td></td></tr><tr><td>3500xL Genetic Analyzer</td><td>Thermo Fisher Scientific</td><td>4405633</td><td></td></tr><tr><td>3500 Series 2 program</td><td>Thermo Fisher Scientific</td><td>4476988</td><td></td></tr><tr><td>Gene Mapper 5 program</td><td>Thermo Fisher Scientific</td><td>4475073</td><td></td></tr><tr><td>Gentra Puregene Cell Kit</td><td>QIAGEN</td><td>1045696</td><td></td></tr><tr><td>Wizard SV Gel and PCR Clean-Up System</td><td>Promega</td><td>A9282</td><td></td></tr><tr><td>NAP1L1 Antibody (N-term)</td><td>Abgent</td><td>AP1920b</td><td></td></tr><tr><td>Nuclear Matrix Protein p84 antibody [5E10]</td><td>GeneTex</td><td>GTX70220</td><td></td></tr><tr><td>Peroxidase AffiniPure Goat Anti-Rabbit IgG</td><td>Jackson ImmunoResearch</td><td>111-035-144</td><td></td></tr><tr><td>Peroxidase AffiniPure Sheep Anti-Mouse IgG</td><td>Jackson ImmunoResearch</td><td>515-035-003</td><td></td></tr></tbody></table>

using a fluorescent pcr-capillary gel electrophoresis technique to genotype crispr/cas9-mediated knockout mutants in a high-throughput format

프로그램 게놈 편집 도구의 개발은 특정 게놈 서열이 세포 전체 유기체의 기능에서하는 역할을 이해하는 역 유전학의 사용을 촉진했다. 이 원인은 엄청나게 CRISPR의 최근 도입 연구자하기 위해, 다른 것들 중, 게놈과 전 사체를 조작 노크, 노크, 또는 표적 유전자에 노크 할 수 있습니다 / Cas9 시스템 - 다용도 도구의 도움되었습니다 방법. 유전자를 녹아웃의 목적을 위해, CRISPR / 이중 가닥 나누기 휴식 사이트에서 뉴클레오티드의 틀 이동을 유발하는 삽입 또는 삭제를 소개하는 비 동종 최종 합류 DNA 수리 경로를 모집 Cas9는 매개. 그러나, 개별 가이드 RNA가 바람직하지 않은 오프 대상 영향을 일으킬 수 있으며, 이들을 배제하기 위해, 여러 가이드의 RNA의 사용이 필요하다. 대상이 또한 다수의 클론 대량 선별이 교대 EF에서의 사용을 돌려서, 이는 요구되는 것을 의미녹아웃 클론 유전자형 높은 처리량 기술을 ficient. 현재 유전자형 기술 중 하나는 따라서 높은 처리량을 위해 적합하지 않다 렌더링, 본질적인 한계로 고통 또는 높은 비용이 발생. 여기서, 주형으로 조 세포 용 해물로부터 게놈 DNA를 사용하여 형광 PCR을 사용하고, 모세관 젤 전기 영동을 통해 PCR 단편을 해결하기위한 우리 상세히 프로토콜. 이 기법은 하나의 단편 간의 염기쌍의 차이를 구별하기에 충분히 정확하고, 따라서 표적 유전자의 코딩 서열의 프레임 이동의 유무를 나타내는에 적합하다. 이 정확한 지식을 효과적으로 확증 시퀀싱 단계의 필요성을 배제하고 사용자가 그 과정에서 시간과 비용을 절약 할 수 있습니다. 여기에 다른 곳에서와 같이 또한,이 기술은 다수의 유전자에 대한 가이드 RNA를 표적 다양한 조직 기원의 다양한 포유 동물 세포 유전형에 다양한 것으로 입증되었습니다.

여기에 설명 된 PCR 형광 모세관 전기 영동 기술은 외래 DNA 전달하는 의무가 거의 모든 세포주에서 유전자 타겟팅 임의 영역에 적용될 것으로 기대된다. 우리는 이전에 대장 암 세포주 12 세 유전자를 대상으로 자사의 응용 프로그램을 증명하고있다. 여기서, 우리는 CRISPR와 대상으로 간암 세포주 인 HepG2를 유전형에서의 효능 / Cas9 1 (NAP1L1) 유전자와 마찬가...

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

형광 중합 효소 연쇄 반응 (PCR)은 겔 전기 영동, 모세관에 결합 여기에 설명 된 기술 유전자형은 뉴 클레아 제 매개 녹아웃 클론의 높은 처리량 유전자형을 허용한다. 그것은 다른 유전자형 기술에 의해 직면 한 한계를 우회하고 시퀀싱 방법보다 더 비용 효과적입니다.

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 ...

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Research

JoVE Journal

Genetics

13.7K 조회수.  Duke-NUS Medical School.  형광 중합 효소 연쇄 반응 (PCR)은 겔 전기 영동, 모세관에 결합 여기에 설명 된 기술 유전자형은 뉴 클레아 제 매개 녹아웃 클론의 높은 처리량 유전자형을 허용한다. 그것은 다른 유전자형 기술에 의해 직면 한 한계를 우회하고 시퀀싱 방법보다 더 비용 효과적입니다. 

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

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

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

포유류 세포에서 CRISPR / Cas9 매개 유전자 knockouts의 선택 의존 및 독립적 생성

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

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

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

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

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

효율적인 생산 및 모델 시스템 Danio rerio 에 유전자 녹아웃 식별의 CRISPR/Cas9 생성

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

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

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

CRISPR/Cas9에 의해 Murine 인간과 조 혈 조상 세포의 고효율 유전자 장애

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

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

빠른 고 손쉬운 파이프라인 C. 선 충 을 사용 하 여 CRISPR/Cas9 Ribonucleoproteins에서 게놈 포인트 돌연변이 생성에 대 한

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

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 ...

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 ...

<meta charset="utf8"></head><body>치료 저항성을 조사하기 위해 CART 세포에서 게놈 와이드 CRISPR 스크리닝

Performing an In Vitro Genome-Wide CRISPR Knockout Screen in Chimeric Antigen Receptor T Cells

Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, the durable response remains limited. Recent studies have shown that the epigenetic landscape of preinfusion CART cell products can influence response to therapy, and gene editing has been proposed as a solution. However, more work needs to be done to determine the optimal gene editing strategy. Genome-wide CRISPR screens have become popular tools to both investigate mechanisms of resistance and optimize gene editing strategies. Yet their application to primary cells presents many challenges. Here we describe a method to complete a genome-wide CRISPR knockout screen in CART cells from healthy donors. As a proof-of-concept model, we designed this method to investigate the development of exhaustion in CART cells targeting the CD19 antigen. However, we believe that this approach can be used to address a variety of mechanisms of resistance to therapy in different CAR constructs and tumor models.

<meta charset="utf8"></head><body>키메라 항원 수용체 T 세포에서 체외 게놈 와이드 CRISPR 녹아웃 스크리닝 수행

The article describes a protocol for the application of an in vitro model for exhaustion to complete a genome-wide CRISPR knockout screen in healthy donor chimeric antigen receptor T cells.

키메라 항원 수용체 T 세포에서 체외 게놈 와이드 CRISPR 녹아웃 스크리닝 수행

Chimeric antigen receptor T (CART) cell therapy is an innovative form of targeted immunotherapy that has revolutionized the treatment of cancer. However, ...

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

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이 비디오의 챕터

포유류 세포에서 CRISPR / Cas9 매개 유전자 knockouts의 선택 의존 및 독립적 생성

효율적인 생산 및 모델 시스템 Danio rerio 에 유전자 녹아웃 식별의 CRISPR/Cas9 생성

CRISPR/Cas9에 의해 Murine 인간과 조 혈 조상 세포의 고효율 유전자 장애

빠른 고 손쉬운 파이프라인 C. 선 충 을 사용 하 여 CRISPR/Cas9 Ribonucleoproteins에서 게놈 포인트 돌연변이 생성에 대 한

포유류 세포에 있는 풀린 CRISPR 기지를 둔 유전 스크린

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

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

키메라 항원 수용체 T 세포에서 체외 게놈 와이드 CRISPR 녹아웃 스크리닝 수행

키메라 항원 수용체 T 세포에서 체외 게놈 와이드 CRISPR 녹아웃 스크리닝 수행

키메라 항원 수용체 T 세포에서 체외 게놈 와이드 CRISPR 녹아웃 스크리닝 수행