crisprcas9技术-完整版
crisprcas9基因编辑技术流程
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基因编辑技术CRISPRCas9的使用教程与最佳实践分享
基因编辑技术CRISPRCas9的使用教程与最佳实践分享在现代生物学研究中，基因编辑技术的出现为研究人员提供了一种高效、精确、低成本的方式来研究基因功能和调控机制。
CRISPR-Cas9系统作为一种革命性的基因编辑工具被广泛应用于基因组编辑、疾病治疗和农业改良等领域。
本文将为您介绍CRISPR-Cas9基因编辑技术的使用教程，并分享一些最佳实践。
CRISPR-Cas9基因编辑技术概述CRISPR-Cas9是一种依靠细菌天然的免疫机制发展而来的基因编辑技术。
CRISPR是一种特殊的DNA序列，可与Cas9酶一起，通过识别和切割DNA序列来精确编辑基因组。
CRISPR-Cas9系统的主要组成部分包括CRISPR RNA （crRNA）、转录单元结构化RNA（tracrRNA）和Cas9酶。
crRNA负责识别目标DNA序列，而tracrRNA将crRNA与Cas9酶结合起来形成活跃的CRISPR-Cas9复合物。
CRISPR-Cas9基因编辑技术的使用教程1. 设计并合成RNA引导序列在使用CRISPR-Cas9进行基因编辑之前，首先需要设计并合成RNA引导序列。
该序列用于指导Cas9酶精确识别和切割目标基因组DNA。
合成的RNA引导序列通常由crRNA和tracrRNA合成而成，也可以合成一个融合的single-guide RNA （sgRNA）。
2. 构建CRISPR-Cas9载体CRISPR-Cas9基因编辑需要将Cas9酶和RNA引导序列导入目标细胞内。
可使用载体如质粒或病毒进行基因编辑构建。
选择合适的载体需考虑目标细胞类型、转染效率和所需编辑范围等因素。
将Cas9基因和RNA引导序列克隆至载体后，可通过转染或病毒介导转染等方法将其导入目标细胞。
3. 确定编辑效果在导入CRISPR-Cas9系统后，使用分子生物学方法来验证编辑效果。
例如，PCR、测序、Western blot或免疫组化等技术可以用于检测目标基因的突变、修复或敲除效果。
基因编辑方法CRISPR-Cas9
构建方案 ： 1. 一是将gRNA 与 Cas9 连入同一个质粒载体中并以双启动子启动，载
体中携带荧光报告基因（用于流式细胞仪筛选）或抗性基因（用于 药物筛选） 。 2. 二是将 gRNA与 Cas9 分别连载不同的载体上，两个载体携带有不同 的荧光报告基因或抗性基因
载体选择：
慢病毒载体 以HIV-1的主要功能元件为基础构建起来的转基因和基
定时间，组织的基因编辑。
CRISPR-Cas9技术的应用进展
1. CRISPR/Cas 系统可以作为一种位点特异性基因编辑平台
Cell 2014 157,1262-1278 Figure2A
Cas9-sgRNA 在靶位点切割产生DSB后， 细胞可以通过两种方式对 DNA 进行修复， 非同源末端连接 （non-homologous endjoining，NHEJ）修 复方式和同源重组方式（homology-directed repair，HDR）。 NHEJ 往往使 得核酸链被剪切的区域发生基因突变，导致编码的基因Knockdown。 而 HDR往往通过供体 DNA 与基因组 DNA 之间的同源重组造成靶位点的纠正 或者靶向插入外源基因Knockin。
利用测序、RFLP 等方法检测基因组定点修饰结果
sgRNA 的设计
• 选择 PAM（NGG）5‘端的一段碱基序列20nt作为原间隔序列，即 敲除的靶位点。(PAM，Protospacer adjacent motifs 原间隔序列临 近基序。一般形式为 NGG，其作用是将间隔序列定位于入侵的噬 菌体或质粒的 DNA 序列中)
Thanks
CRISPR-Cas9技术的应用进展
2. 利用 Cas9 系统在转录水平对基因表达的调节
Cell 2014 157,1262-1278 Figure6G
Thermo Fisher Scientific Inc. CRISPR-Cas9基因组编辑技术说明
12 | /bioprobes© 2016 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.CrIsPr-CAs9–BAsED rEsEArCH BIOPrOBEs 74The CRISPR-Cas9 system for genome editingA complete suite of reagents, from Cas9 delivery tools to cell function assays.The transformative CRISPR-Cas9 technology is revolutionizing the field of genome editing. Derived from components of an adaptive immune system in bacteria, the CRISPR-Cas9 system enables targeted gene cleavage and gene editing in a wide variety of eukaryotic cells. Because the specificity of the endonuclease cleavage is guided by RNA sequences, editing can be directed to virtually any genomic locus simply by engineering the guide RNA sequence and delivering it along with the Cas endonuclease to the target cell. The CRISPR-Cas9 system has great promise in broad applications such as stem cell engineering, gene therapy, tissue and animal disease models, and the development of disease-resistant transgenic plants.The CRISPR-Cas9 system derives its specificity from a short, noncoding guide RNA (gRNA) that has two molecular components: a target-specific CRISPR RNA (crRNA) and an auxiliary trans-activating CRISPR RNA (tracrRNA). The gRNA guides the Cas9 protein to a specific genomic locus via base pairing with the target sequence (Figure 1). Upon binding to the target sequence, the Cas9 protein induces a specific double-strand break. Following DNA cleavage, the break is repaired by cellular repair machinery through nonhomologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms. Withtarget specificity defined by a very short RNA sequence coupled with an efficient endonuclease activity, the CRISPR-Cas9 system greatly simplifies directed genome editing.Choose the right CRISPR-Cas9 delivery methodSeveral strategies are available for delivering Cas9 protein to target cells,and this flexibility is one of the key advantages when using CRISPR-Cas9 genome editing technology in different experimental systems (Figure 2). Advances in DNA, mRNA, and protein delivery methods have significantly streamlined the process, making the introduction of Cas9 more efficient and with minimal off-target effects. Thermo Fisher Scientific offers four formats for CRISPR-Cas9 delivery: Invitrogen ™ GeneArt ™ CRISPR Nuclease Vector (DNA), GeneArt ™ CRISPR NucleaseFigure 1. A CRISPR-Cas9 targeted double-strand break. Cas9-mediated cleav-age occurs on both strands of the DNA, three base pairs upstream of the NGG proto-spacer adjacent motif (PAM) sequence on the 3’ end of the target sequence. The specificity is supplied by the guide RNA (gRNA), and changing the target only requires a change in the design of the sequence encoding the gRNA. After the gRNA unit has guided the Cas9 nuclease to a specific genomic locus, the Cas9 protein induces a double-strand break at the specific genomic target sequence.Figure 2. Options for efficient CRISPR-Cas9 delivery. In the DNA delivery format, the CRISPR DNA vector enters the cell and translocates to the nucleus, where the Cas9 mRNA and gRNA are transcribed. Translated in the cytoplasm, the Cas9 protein combines with the gRNA to form a ribonucleoprotein (RNP) complex that then enters the nucleus for targeted gene editing. In the RNA delivery format, the Cas9 mRNA and gRNA are cotransfected into the cell cytoplasm, where the mRNA is translated to produce functional Cas9 protein. The Cas9-gRNA (RNP) protein delivery format streamlines cell engineering by eliminating transcription and translation in the cell and produces the highest cleavage efficiencies in our labs. With the RNP format there is no requirement for a specific promoter, nor concern over random integration into the genome; the Cas9 RNP complex can act immediately after it enters the cell, since transcription and translation are not required. Moreover, the complex is rapidly cleared from the cell, minimizing the chance of off-target cleavage events when compared to vector-based systems.Cas9native sequence results in frameshifts or mutations, gene tags, and single or multiple genesCotransfect cells with donor DNA/bioprobes |13 © 2016 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.BIOPrOBEs 74 CrIsPr-CAs9–BAsED rEsEArCHdirectly, and when antibiotic selection is used to identify transfected cells, viability assays can be used to monitor the selection process.Monitoring the efficiency of genome editing. When using genome editing tools—such as CRISPR-Cas9, TAL effectors, or zinc finger nucleases—to obtain targeted mutations, you need to determine the efficiency with which these nucleases cleave the target sequence, prior to continuing with labor-intensive and expensive experiments. The Invitrogen ™ GeneArt ™ Genomic Cleavage Detection Kit provides a simple and reliable assay for the cleavage efficiency of genome editing tools at a given locus. In this assay, a sample of the edited cell population is used as a direct PCR template for amplification with primers specific to the targeted region. The PCR product is then denatured and reannealed to produce heteroduplex mismatches where double-strand breaks have occurred, resulting in insertion/deletion (indel) introduction. These mismatches are recognized and cleaved by the detection enzyme, and the cleavage is easily detectable and quantifiable by gel analysis.Cell phenotyping. The CRISPR-Cas9 system is routinely used for knockout, knock-in, or modulation of gene expression, and the primaryon-target effects can be measured using cell analysis techniques; west-ern blotting, flow cytometry, and fluorescence microscopy are often used to view changes to protein expression or structure in a cell population. Flow cytometry provides the throughput for multiparameter analysis on vast numbers of individual cells. Cell imaging (Figure 4) allows for direct analysis of changes in protein expression, compartmentalization, and cell morphology; high-content analysis (HCA) provides automation for the imaging process coupled with quantitative rigor.mRNA (mRNA), GeneArt ™ Platinum ™ Cas9 Nuclease (protein) (Figure 2), and CRISPR library services (see page 11). Based on the cell type and application, the most effective delivery format can be chosen and then paired with optimal cell culture reagents and analysis tools.Monitor the genome editing process from start to finishWhichever CRISPR-Cas9 delivery strategy you choose, it is important to carefully monitor the entire genome editing process to validate that Cas9 protein has been successfully incorporated into cells and that the target knockout or mutation has been accurately implemented. This monitoring can be broken down into four categories:Cell culture. The starting point for genome editing is healthy cells. Performing cell health assays prior to using the CRISPR-Cas9 system can serve as an important quality control step and help to avoid wasting time and reagents. Tests for viability, apoptosis, and stress responses should be a routine part of cell growth and can provide information to optimize experimental conditions to produce the most robust cells.Genome editing. Immunochemical assays such as western blots can effectively measure the presence of Cas9 in cells. Figure 3 shows that the accumulation of Cas9 protein varies considerably depending on the choice of delivery method (plasmid, mRNA, or protein). Together with immunocytochemistry, antibiotic selection and gene expression are frequently used to monitor the assembly of CRISPR components for gene editing in the cell. Fluorescent protein expression can be measuredFigure 3. Western blot detection of Cas9 accumulation over time in cells transfected with Cas9-expressing plasmid DNA, Cas9 mRNA, or Cas9 protein. HEK293FT cells were transfected with Cas9 plasmid DNA, mRNA, or protein and then harvested at indicated times for western blot analysis. Proteins in the cell lysates were separated on an Invitrogen ™ NuPAGE ™ Novex ™ 4–12% Bis-Tris Protein Gel, transferred to a PVDF membrane using the Invitrogen ™ iBlot ™ 2 Gel Transfer Device, and incubated with an anti-Cas9 mouse monoclonal antibody at 1:3,000 dilution and an HRP-conjugated rabbit anti–mouse IgG antibody at 1:2,000. The membrane was developed using Thermo Scientific ™ Pierce ™ ECL Western Blotting Substrate (Cat. No. 32106). Reprinted with permission from Liang X, Potter J, Kumar S et al. (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol 208:44–53.DNA 72ProteinmRNA 4824840(hr)Time Figure 4. Absence of LC3B in CRISPR-Cas9–edited HAP1 cells after chloro-quine treatment. HAP1 cells were modified using CRISPR-Cas9 gene editing to knock out the ATG5 gene. After chloroquine treatment, which normally causes LC3-containing autophagosomes to accumulate, edited cells (right panel) show the expected absence of LC3B-positive puncta, whereas wild-type cells (left panel) show an increase in LC3B accumulation. Cells were labeled with rabbit anti-LC3B antibody (Cat. No. L10382) followed by Invitrogen ™ Alexa Fluor ™ 647 goat anti–rabbit IgG antibody (red, Cat. No. A21245) and counterstained with Hoechst ™ 33342 (blue, Cat. No. H3570). Images were acquired on a Thermo Scientific ™ CellInsight ™ CX5High-Content Screening (HCS) Platform.14 | /bioprobes© 2016 Thermo Fisher Scientific Inc. All rights reserved. For Research Use Only. Not for use in diagnostic procedures.CrIsPr-CAs9–BAsED rEsEArCH BIOPrOBEs 74Product Quantity Cat. No.CRISPR proteinGeneArt ™ Platinum Cas9 Nuclease (1 µg/µL)GeneArt ™ Platinum Cas9 Nuclease (1 µg/µL)GeneArt ™ Platinum Cas9 Nuclease (3 µg/µL)10 µg 25 µg 75 µg B25642B25640B25641CRISPR mRNAGeneArt ™ CRISPR Nuclease mRNA 15 µg A29378GeneArt ™ Strings U6 DNA >200 ng **************************************GeneArt ™ Strings T7 DNA >200 ng **************************************Custom in vitro –transcribed gRNA 250 nmol ***********************************CRISPR plasmidGeneArt ™ CRISPR Nuclease Vector with OFP Reporter Kit10 reactions A21174GeneArt ™ CRISPR Nuclease Vector with OFP Reporter, with competent cells 10 reactions A21178GeneArt ™ CRISPR Nuclease Vector with CD4 Enrichment Kit10 reactions A21175GeneArt ™ CRISPR Nuclease Vector with CD4 Enrichment Kit, with competent cells 10 reactionsA21177CRISPR-Cas9 gRNAGeneArt ™ Precision gRNA Synthesis KitA29377CRISPR libraries: see page 11 and go to /crisprlibraries .Forcustom(arrayedorpooled)CRISPRlibraries,***********************************.CRISPR engineered cell lines: go to /engineeredcells .Forcustomstablecelllinegenerationservices,***********************************.Detection and analysis reagents GeneArt ™ Genomic Cleavage Detection Kit GeneArt ™ Genomic Cleavage Selection Kit20 reactions 10 reactionsA24372A27663Figure 5. Rapid analysis of various cell health parameters using a high-content analysis (HCA) platform. Wild-type and CRISPR-edited HAP1 cells were analyzed using the Thermo Scientific ™ CellInsight ™ CX5 High-Content Screening Platform for (A) apoptosis, using CellEvent ™ Caspase-3/7 Green Detection Reagent (Cat. No. R37111), (B) oxidative stress, using CellROX ™ Green Reagent (Cat. No. C10444), (C) protein degradation, with the Click-iT ™ HPG Alexa Fluor ™ 488 Protein Synthesis Assay Kit (Cat. No. C10428), and (D) protein synthesis, using the Click-iT ™ Plus OPP Alexa Fluor ™ 488 Protein Synthesis Assay Kit (Cat. No. C10456).ABCChloroquine concentration (μM)M e a n c i r c a v g i n t e n s i t yMenadione concentration (μM)M e a n c i r c a v g i n t e n s it yStaurosporine concentration (μM)M e a n c i r c a v g i n t e n s i t yDCycloheximide concentration (μM)M e a n c i r c a v g i n t e n s i t yWith modulation of any cellular signaling pathway comes the risk of proximal and distal consequences. It is important to track your targeted protein and also monitor the impact on other aspects of cell health and behavior (off-target phenotyping). HCA is particularly suited to this type of multiparameter investigation (Figure 5).Resources to help you get startedThermo Fisher Scientific offers a wide range of reagents, kits, and tools to support your genome editing experiments (Table 1). In addi-tion to our state-of-the-art online Invitrogen ™ CRISPR Search and Design Tool, we offer several different Cas9 delivery systems as well as cell culture reagents and cell analysis tools that can be matched to your experimental system. Our suite of genome editing products isTable 1. Online CRISPR-Cas9 resources from Thermo Fisher Scientific.selection guide Delivery formatproduct selection guide /genomeedit101gRNA design tool /crisprdesign Products for monitoring genome editing/detectcrisprcontinually expanding to include the entire cell engineering workflow, from reagents for cell culture, transfection, and sample preparation to kits for genome modification and for detection and analysis of known genetic variants. Go to /detectcrisprbp74 for an up-to-date view of our products and technologies. ■。
CRISPRCas9基因编辑操作步骤及详细说明
CRISPRCas9基因编辑操作步骤及详细说明实验材料与方法一、细胞培养人宫颈癌细胞 HeLa，常规培养使用含 10% FBS 的 DMEM 培养基 ( 含 1.5 mg/L-Glutamine,100 U/mL Penicillin,100 μg/mL Streptomycin) 中，37ºC 5% CO2 饱和湿度培养箱中培养。
二、基因信息及双 gRNA 设计基因信息及分析1.hsa-mir-152 基因信息：pubmed2.hsa-mir-152 基因位于蛋白编码基因 COPZ2 内含子内，敲除hsa-mir-152 基因不会影响该蛋白编码3.hsa-mir-152 precursor 序列（87 bp）：TGTCCCCCCCGGCCCAGGTTCTGTGATACACTCCGACTCGGGCTCTGGAGCAGTCAGTGCATGACAGAACTTGGGCCCGGAAGGACC双 gRNA 设计使用在线 gRNA 设计软件在 hsa-mir-152 precursor 基因组序列两侧设计双 gRNA注：dgRNA 即为双 gRNA.三、慢病毒侵染实验材料及试剂DMEM 培养基 + 10% FBSD-Hank’s SolutionTrypsin-EDTA Solution96 孔板24 孔板Lentivirus- 病毒液（GenePharma）步骤靶细胞侵染实验1.靶细胞铺板：24-well，加入2.5×105 cells/well（根据细胞种类调整），0.5 mL 完全培养基，37℃，5% CO2 过夜；2.稀释病毒：稀释液（靶细胞维持液培养基）400 μL + 终浓度 5 μg/mL Polybrene，将慢病毒原液按 1:9 加入到稀释液中；3.移去 Step1 中细胞培养液，加入 Step2 稀释后的病毒液，同时建立对照（blank、negative），37℃，5% CO2 过夜；4.12~24 小时移去细胞侵染后的病毒液，加入 0.5 mL 完全培养基，37℃，5% CO2 过夜；5.根据细胞状态和类型，如果必要分出 1/3~1/5，加入0.5 mL 完全培养基，继续培养 24~48 小时，荧光倒置显微镜下观察结果。
Biomics_CRISPR_Cas9_基因敲除技术
CRISPR/Cas9 基因敲除技术CRISPR （ClusteredRegularlyInterspacedShortPalindromicRepeats）RNA，是最近几年才发现的原核生物中的调控RNA，用以抵御病毒和质粒入侵。
在II型CRISPR系统中，CRISPR RNA（crRNA）与转录激活crRNA（Trans-activating crRNA, tracrRNA）退火形成的复合物能特异识别基因组序列，引导Cas9核酸内切酶在目的片段生成DNA双链断裂（double-strand breaks, DSBs）。
CRISPR-Cas系统的高效基因组编辑功能已被应用于多种生物，包括人、小鼠、大鼠、斑马鱼、秀丽隐杆线虫、植物及细菌。
多个科研小组的研究都显示，与锌指核酸酶（ZFNs）和转录激活样效应核酸酶（Transcription activator-like effector nucleases, TALEN）相比较，CRISPR-Cas系统介导的基因组靶向实验在真核细胞中具有相似甚至更高的效率。
Biomics专注于RNA基因调控多年，最新推出基因组编辑工具CRISPR/Cas9专家系统，该系统灵活简单、可以对特定基因组位点进行切割置换，特异性高、细胞毒性低。
CRISPR/Cas9系统可广泛应用于基因组工程，如基因抑制，基因敲除，基因敲入，基因修复等。
CRISPR-Cas9体系的RNA-DNA识别机制为基因组工程研究提供了一项简便而强大的工具。
其最重要的优势是Cas9蛋白可在多个不同的gRNA的引导下同时靶向多个基因组位点，起到多靶点调控的作用。
z CRISPR与RNAi的区别目前已经广泛应用的RNAi技术的靶标是mRNA，而CRISPR通过RNA识别DNA序列然后再改变DNA序列，是可以遗传的。
由于编码mRNA的DNA序列只占总DNA的极少部分，因此靶向DNA序列的CRISPR的靶标要比RNAi广得多，更有可能筛选出针对某DNA序列的特异CRISPR靶标。
crispr-cas9技术操作流程
crispr-cas9技术操作流程下载温馨提示:该文档是我店铺精心编制而成，希望大家下载以后，能够帮助大家解决实际的问题。
文档下载后可定制随意修改，请根据实际需要进行相应的调整和使用，谢谢!并且，本店铺为大家提供各种各样类型的实用资料，如教育随笔、日记赏析、句子摘抄、古诗大全、经典美文、话题作文、工作总结、词语解析、文案摘录、其他资料等等，如想了解不同资料格式和写法，敬请关注!Download tips: This document is carefully compiled by theeditor.I hope that after you download them,they can help yousolve practical problems. The document can be customized andmodified after downloading,please adjust and use it according toactual needs, thank you!In addition, our shop provides you with various types ofpractical materials,such as educational essays, diaryappreciation,sentence excerpts,ancient poems,classic articles,topic composition,work summary,word parsing,copy excerpts,other materials and so on,want to know different data formats andwriting methods,please pay attention!CRISPR-Cas9技术的操作流程详解CRISPR-Cas9，全称为“簇状规律间隔短回文重复序列-CRISPR相关蛋白9”，是一种革命性的基因编辑工具，它使得科学家能够精确地添加、删除或修改DNA序列。
crispr cas9技术
1.2 .CRISPR/Cas9作用机理
CRISPR-Cas系统的结构：CRISPR-CAS 系统的组成主要包括: 由 不连续的重复序列R( repeat) 与长度相似的间区序列 S( spacers) 间隔排列而成的CRISPR 簇，前导序列L( leader) 以及一系列CRISPR 相关蛋白基因cas。
CRISPR/cas9基因编辑技术及应 用
——— 一种来源于细菌获得 性免疫由RNA指导Cas蛋白对靶 向基因获得性修饰的技术
1. CRISPR-Cas系统简介
• 1987 年，日本课题组在K12 大肠杆菌的 碱性磷酸酶基因附近发现串联间隔重复序 列，随后发现其广泛存在于细菌和古细菌 的基因组中，2002 年，正式将其命名为 成簇的规律间隔的短回文重复序列 (CRISPR)
在一起得到sgRNA（singleguideRNA）。融合的RNA具有与
野生型RNA类似的活力，但因为结构得到了简化更方便研究者使
用。通过将表达sgRNA的原件与表达Cas9的原件相连接，得到可
以同时表达两者的质粒，将其转染细胞，便能够对目的基因进行
操 Ca作s9技术介导单基因细胞系敲除的实验流程（Cas9质粒建立 k1n、oc设k-计ou识t细别靶胞位系点）的一对DNA Oligos
CRISPR/技术的前景及优缺点
1CRISPR-Cas9技术的优势
而且从实际应用的角度来说，CRISPRs比TALENs更容易操作，因为 每一对TALENs都需要重新合成，而用于CRISPR的gRNA只需要替换 20个核苷酸就行。