Cas9 protocol_v1.1_130216

CRISPR-Cas9植物基因敲除试剂盒说明书Cat. No. GP0138Protocol No. PT140730-1出版日期July.2014南京市汉中门大街301号南京国际服务外包产业园01栋13层A座电话：+86-25-66776700/66776718传真：+86-25-66776701邮编：210036网址：CRISPR-Cas9植物基因敲除试剂盒目录I．组份列表 (2)II．产品概要 (2)III．操作步骤 (3)1. 设计Oligo DNA 序列 (3)2. 线性化pP1C.2载体 (3)3. 重组pP1C.2载体的构建 (3)4. 重组pP1C.1载体的构建 (3)IV．附录pP1C系统质粒图谱..................................................................................4-5 V．参考文献. (6)图Figure 1. CRISPR-Cas9工作原理示意图Figure 2. pP1C.1质粒图谱Figure 3. pP1C.2质粒图谱表Table1. CRISPR-Cas9植物基因敲除试剂盒组份列表Protocol No. PT140730-1 Genloci Biotechnologies Inc.1/6CRISPR-Cas9植物基因敲除试剂盒I．组份列表CRISPR-Cas9基因敲除试剂盒组份如下：Table1. CRISPR-Cas9植物基因敲除试剂盒组份列表组份列表含量pP1C.1 Vector2μgpP1C.2 Vector 2μg贮存条件※注意：在收到货后，请您将pP1C.1 Vector 和pP1C.2 Vector瞬时离心后再保存。

将pP1C.1 Vector、pP1C.2 Vector置于-20℃保存，且避免污染；II．产品概要CRISPR/Cas9植物基因敲除试剂盒是一款高效、精准的基因编辑试剂盒，它是基于最新代的人工核酸内切酶CRISPR-Cas9而研发的，相对于传统敲除技术和TALENs\ZFNs技术而言，其操作更加简便，敲除效率最高，基因的编辑更加的精准，大大降低了脱靶机率。

crispr-cas9进行全基因组的筛选Human CRISPR Knockout Pooled Library (Brunello) 一、lentiCas9-Blast质粒的基本信息：/pooled-library/broadgpp-human-knockout-b runello/由于cas9自待的抗性基因是我们不需要，在抗性基因两边突变出酶切位点，酶切掉原本的抗性基因。

换成另一种。

1基。

2向离心管中加入质粒DNA和等体积的Opti-MEM，混匀，调整总体积为2.5 ml，在室温下温育5分钟。

3将Lipofectamine 2000试剂轻柔摇匀，取100 μl Lipofectamine 2000试剂在另一管中与2.4 ml Opti-MEM混合，在室温下温育5分钟。

4把稀释后的DNA与稀释后的Lipofectamine 2000进行混合，轻轻地颠倒混匀，不要振荡。

必须在5分钟之内混合。

5混合后，在室温下温育20分钟，以便形成DNA 与Lipofectamine 2000稀释液的转染复合物。

6DNA与Lipofectamine 2000混合液转移至293T细胞的培养液中，混匀，于37 ℃, 5% CO2 细胞培养箱中培养。

7培养8 h后倒去含有转染混和物的培养基，每瓶细胞加入20 ml的PBS液，轻轻左右晃动一下培养瓶以洗涤残余的转染混和物，然后倒去。

8每瓶细胞中加入含10% 血清的细胞培养基25 ml，于37 ℃、5% CO2培养箱内继续培养48小时。

9收集转染后48小时（转染即可为0小时计起）的293T细胞上清液。

10于4 ℃，4000 g 离心10 min，除去细胞碎片。

11以0.45 μm滤器过滤上清液于15ml 离心管中。

二、慢病毒转染细胞参考网址：/protocols/generatin g-stable-cell-lines/1. 取六孔板铺50000个细胞， 37°C,5% CO 2 培养，待细胞长到70%，换含有8ug/ml polybrene的培养基培养。

cas9 构建基因敲除细胞系步骤1. 设计gRNA序列需要设计合适的gRNA（guide RNA）序列。

gRNA是一种由RNA和DNA组成的分子，在CRISPR-Cas9系统中起到引导Cas9蛋白识别和切割目标DNA的作用。

gRNA序列应当与目标基因的特定区域互补，并且要避免与其他基因的序列相互匹配。

2. 合成gRNA和Cas9蛋白合成设计好的gRNA序列，并将其与Cas9蛋白结合。

Cas9蛋白是CRISPR-Cas9系统中的核酸酶，能够与gRNA一起识别目标DNA 序列，并在其靶向区域引发双链断裂。

3. 转染细胞将gRNA和Cas9蛋白复合物转染到目标细胞中。

转染可以使用多种方法，如化学法、电穿孔法或病毒载体介导的转染等。

转染后，gRNA和Cas9蛋白会进入细胞质，并寻找目标基因。

4. 筛选敲除细胞系根据需要敲除的基因，选择适当的筛选方法来鉴定敲除细胞系。

常用的筛选方法包括PCR、Western blot和细胞克隆等。

通过这些方法，可以检测到目标基因的敲除情况。

5. 确认敲除细胞系对筛选出的细胞系进行进一步的确认。

可以使用DNA测序技术对目标基因的敲除位点进行测序验证。

此外，还可以通过功能实验来验证目标基因的敲除效果。

6. 存储和维护敲除细胞系成功敲除目标基因后，需要对敲除细胞系进行存储和维护。

细胞系应当保存在液氮中，以确保其长期保存和使用的稳定性。

同时，细胞系也需要定期培养和检测，以确保其生长状态和敲除效果。

以cas9构建基因敲除细胞系的步骤包括设计gRNA序列、合成gRNA和Cas9蛋白、转染细胞、筛选敲除细胞系、确认敲除细胞系以及存储和维护敲除细胞系。

这些步骤需要在实验室中进行，确保操作的准确性和稳定性。

基因敲除细胞系的建立为研究基因功能和疾病机制提供了有力的工具，对于深入理解生命活动和疾病发生发展具有重要意义。

CRISPR/Cas9-based genome editing technologyA robust CRISPR/Cas9 vector system for multiplex targeting ofgenomic sites in monocot and dicot plants亚热带农业生物资源保护与利用国家重点实验室华南农业大学生命科学学院刘耀光课题组(**************.cn )1. pYLCRISPR/Cas9多靶点载体介绍CRISPR/Cas9是新近发展的基因组编辑技术（图1）。

CRISPR/Cas9切割靶序列仅需要single-guide RNA (sgRNA)以及由sgRNA 引导的Cas9蛋白，比锌指核酸酶（ZFNs ），TALENs 更加简便，高效，因而成为基因组编辑工具的首选。

我们利用CRISPR/Cas9技术可方便地进行多重靶向的特征，构建了一套用于单子叶和双子叶植物的多靶点CRISPR/Cas9基因打靶载体系统。

本套载体将Cas9蛋白表达盒整合到双元载体上，用于装载多个sgRNA 表达盒的多克隆位点Bsa I 位于靠近双元载体RB 位置。

sgRNA 表达盒元件设在中间质粒载体上，利用酶切连接和PCR 方法拼装好，再利用Golden Gate 或Gibson Assembly 克隆方法组装到双元载体上。

本载体系统已经发表（Ma et al., 2015, Molecular Plant , DOI:10.1016/j.molp.2015.04.007）。

5’ NNNNNNNNNNNN GNNNNNNNNNNNNNNNNNNN Target SitePAM NNNNNNNNNNNN-3’3’ NNNNNNNNNNNN NCCNNNNNNNNNNNN-5’NNNNNNNNNNNNNNNNNNNN 5’-G/A NNNNNNNNNNNNNNNNNNNN GUUUUAGAGCUAG A A CGAUA GAAAACUAUUGCCUGAUCGGAAUAAAAUU Cas9nuclease Cleavage site genomesequenceRuvC-like domain HNH domain CUUGAAAAAGUGGCACCGA G CGUGGCU UUUUU-3’NGGFigure 1. A working model of the CRISPR/Cas9 system. The Cas9-sgRNA complex locates to the target site to cleave the DNA to produce double strand break (DSB).2．CRISPR/Cas9载体与sgRNA载体图谱2.1CRISPR/Cas9双元载体本套载体系统的双元载体骨架为pCAMBIA-1300 (ACCESSION: AF234296)，Cas9p为本实验室设计合成的植物优化密码子基因，它模拟了禾本科植物基因具有5’端GC含量较高的特征(Figure 2)。

CRISPRCas9基因敲除细胞株详细构建流程Puro 抗性浓度摸索实验将细胞如下图1稀释。

给药7天后，弃培养液，用台盼蓝染色2 min，显微镜下观察细胞存活情况。

确定细胞多克隆细胞筛选和单克隆细胞筛选浓度。

图1 抗性浓度摸索单克隆形成验证实验细胞计数，将细胞悬液稀释混匀后加入96孔板，用封口胶将孔板封好，放于培养箱中培养。

静置培养48 h后每日观察并记录单克隆形成情况。

sgRNA 靶标设计根据基因序列信息，设计 sgRNA。

靶标位点序列信息确认PCR扩增（图2），测序验证基因序列，以确定sgRNA区域有无SNP。

图2 靶标序列扩增sgRNA克隆引物合成根据sgRNA设计sgRNA克隆引物。

lentiCRISPRv2-sgRNA载体构建退火，连接，转化，涂板（LB/Amp）培养。

lentiCRISPRv2-sgRNA载体验证每个实验组各挑取6个单克隆菌落，于LB/ Amp培养基中扩增，提取质粒，琼脂糖凝胶电泳检测质粒抽提效果（图3）。

图3 质粒抽提酶切验证：取3个单克隆进行酶切，琼脂糖凝胶电泳检测酶切效果（图4）。

选择2个样品送样测序。

图4 单克隆酶切验证病毒包装lentiCRISPRv2-sgRNA无内毒素质粒提取，病毒包装。

细胞转染配制梯度病毒稀释液，细胞于培养箱中静置培养48h。

阳性单克隆细胞株筛选细胞转染48h后，更换完全培养基，筛选至对照组大部分细胞死亡，实验组细胞扩大培养，进行单克隆筛选。

几天后挑选阳性单克隆进行扩增，并取样验证。

阳性单克隆细胞株验证测序验证阳性单克隆细胞株的基因序列，以确定是否敲除成功。

实验结果示例：该基因有两个单克隆细胞株，A1和A2。

单克隆细胞A1的目的基因在sgRNA2位置出现两种突变形式，分别缺失1个和19个碱基，在新序列的第337位和第355位碱基提前出现终止密码子。

单克隆细胞A2的目的基因在sgRNA2位置发生突变，插入1个碱基，在新序列的第340位碱基提前出现终止密码子。

Cas9：一种高效的基因编辑工具Cas9的结构和功能•Cas9是一种核酸酶，能够识别并切割特定的DNA序列•Cas9需要与一条小导向RNA（sgRNA）结合，形成Cas9-sgRNA复合体•sgRNA包含一个与Cas9结合的骨架序列和一个与目标DNA互补的20个碱基的指导序列•Cas9-sgRNA复合体能够识别并结合目标DNA上的一个叫做原型相容性重复序列（PAM）的特殊序列•PAM通常是NGG或NAG，其中N代表任意碱基，G代表鸟嘌呤•Cas9在PAM的上游位置对DNA进行双链切割，形成DNA双链断裂（DSB）Cas9引发的DNA修复机制•DNA双链断裂是一种严重的细胞损伤，细胞会启动两种主要的修复机制来修复DSB：非同源末端连接（NHEJ）和同源重组（HR）•NHEJ是一种高效但不精确的修复方式，它直接将断裂的两个末端连接起来，但在连接过程中可能会发生碱基插入或缺失，导致移码突变•HR是一种精确但低效的修复方式，它需要有一个与目标DNA同源的供体模板，通过拷贝模板上的序列来修复DSB，但在拷贝过程中可能会发生基因转换或交叉互换•Cas9引发的DSB通常会被NHEJ修复，从而实现基因敲除或敲降Cas9 nickase：一种提高特异性的变体•Cas9 nickase是一种经过突变后只能对DNA单链进行切割造成DNA缺口（nicks）的Cas9变体•Cas9 nickase需要与两条sgRNA结合，分别靶向DNA互补的两条链，并在相距10~20个碱基的位置形成两个缺口•两个缺口之间的DNA片段会被切除，形成一个DSB•Cas9 nickase引发的DSB也会被NHEJ修复，从而实现基因敲除或敲降•Cas9 nickase相比于Cas9有更高的特异性和更低的脱靶率，因为它需要同时满足两条sgRNA和两个PAM的匹配条件Cas9在基因编辑中的应用•Cas9是一种强大而灵活的基因编辑工具，它可以实现多种基因操作，如基因敲除、敲降、插入、替换、激活、抑制等•Cas9可以用于研究基因功能、调控基因表达、纠正遗传病、改善农作物、开发新药等领域•Cas9还可以与其他分子工具结合，如转录因子、表观遗传修饰酶、荧光蛋白等，实现更多样化和精细化的基因编辑。

GenCrispr Cas9 Nuclease Cat. No. Z03386 Version 01202016I Description (1)II Kit Contents .......................................................................... .... .... (1)III Key Features ............................... .... .... .... .... .... . (1)IV Storage..................................................................... . (2)V Diluent Compatibility (2)VI Activity test (2)VII References (2)I. DESCRIPTIONGenCrispr Cas9 Nuclease is the recombinant Streptococcus pyogenes Cas9 (wt) protein purified from E. coli that can be used for genome editing by inducing site-specific double stranded breaks in double stranded DNA. Cas9 protein forms a very stable ribonucleoprotein (RNP) complex with the guide RNA (gRNA) component of the CRISPR/Cas9 system. The RNP complex recognizes the target site by matching gRNA with the genomic DNA sequence and produces DNA breaks within 3 bases from the NGG PAM (Protospacer Adjacent Motif). With GenCrispr Cas9 nuclease, customers can screen for highly efficient gRNA in vitro using DNA cleavage assays. The high purity Cas9 protein can also be used for antibody production.Product Source: GenCrispr Cas9 Nuclease is produced by expression in an E. coli strain carrying a plasmid encoding the Cas9 gene from Streptococcus pyogenes without nuclear localization signal (NLS).II. KIT CONTENTSIII. KEY FEATURESHigh Protein Purity: GenCrispr Cas9 Nuclease is > 95% pure as determined by SDS-PAGE with Coomassie Blue detection.Non-specific DNase Activity: A 20 µl reaction in Cas9 reaction buffer containing 100 ng linearized pUC57 plasmid and 0.1 µg of GenCrispr Cas9, incubated for 16 h at 37°C. No DNA degradation is determined by agarose gel electrophoresis.Non-specific RNase Activity: A 10 µl reaction in Cas9 reaction buffer containing 1800 ng total RNA and 0.1 µg of GenCrispr Cas9 incubated for 2h at 37°C. No RNA degradation as determined by agarose gel electrophoresis.High Bioactivity: 20 nM GenCrispr Cas9 incubated for 1 hour at 37°C result in 90% digestion of the substrate DNA as determined by agarose gel electrophoresis.IV. STORAGEGenCrispr Cas9 is supplied with 1x storage buffer (10 mM Tris, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% Glycerol pH 7.4 @ 25°C). The recommended storage temperature is -20°C.V. Diluent CompatibilityDiluent Buffer: 300 mM NaCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, 500 μg/ml BSA and 50% glycerol. (pH 7.4 @ 25°C).VI. Activity testCas9 site-specific digestion:Genscript used in vitro digestion of a linearized plasmid to determine the activity of the Cas9 nuclease. It is a sensitive assay for GenCrispr Cas9 quality control. The linearized plasmid containing the target site:(CATCATTGGAAAACGTTCTT)can be digested with gRNA:(CAUCAUUGGAAAACGUUCUUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUUU)and GenCrispr Cas9. Two cleavage DNA fragments (812 bp and 1898 bp) are determined by agarose gel electrophoresis. A 20 µl reaction in 1xCas9 Nuclease Reaction Buffer containing 160 ng linearized plasmid, 40 nM gRNA and 20 nM GenCrispr Cas9 for 2 hours at 37°C results in 90% digestion of linearized plasmid as determined by agarose gel electrophoresis.VII References1. Jinek et al. A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. (2012)Science 337 (6096) 816-821 (2012).2. Larson, M. H., et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression.NatureProtocols. 8, (11), 2180-2196 (2013).3. Ran, F. A., et al. Genome engineering using the CRISPR-Cas9 system. Nature Protocols. 8, (11), 2281-2308(2013).Notes:1. This is a basic protocol. The reagent concentrations, conditions, and parameters may need to be optimized.2. 1000 nM is equal to 160 ng/µl.GenScript US860 Centennial Ave., Piscataway, NJ 08854Tel: 732-885-9188, 732-885-9688Fax: 732-210-0262, 732-885-5878Email: *********************Web: For Research Use Only.。

CRISPR/Cas9敲除细胞系构建步骤及方法一、技术简介CRISPR/Cas9是最新出现的一种由RNA指导的Cas9核酸酶对靶向基因进行编辑的技术，也是目前研究最热的基因编辑技术。

由于其具有构建方法简单快捷、基因修饰效率高、成本低廉、实验周期短、适用范围广等诸多优点，目前已成功应用于人类细胞、斑马鱼、大/小鼠等多种动植物的基因组精确修饰。

二、实验流程1. 预实验1.1 Cas9导入细胞方法：尝试各种方法，如脂质体类转染、电转、慢病毒感染、腺病毒感染等，确定高效导入Cas9方法。

1.2 药物浓度预实验：降低后续阳性克隆筛选和检测工作难度。

1.3 单克隆培养情况：确认细胞是否可以单克隆培养。

2. 基因敲除（敲入）2.1 靶点设计：一般在不同转录产物的共同外显子上设计3个靶点，靶点位置尽量在基因CDS的前1/3，ATG之后，最好能破坏重要的domain和所有的转录产物isoform。

第一批合成构建3个，效果不佳或时间紧张的可一次构建6个。

2.2 载体构建和病毒包装：根据预实验结果，选择合适的普通载体或病毒载体（普通Cas9载体、慢病毒Cas9载体和腺病毒Cas9载体）。

2.3 内源活性筛选：转染细胞或感染细胞48h后，使用Puro或Blasticidin筛选48h，提取基因组DNA。

使用T7E1酶验证打靶载体的活性，将有效的突变型PCR产物测序验证。

2.4 Donor载体（基因敲入）：根据筛选的gRNA靶点位置，构建Donor普通载体或腺病毒载体，共转染/感染Cas9-gRNA和Donor。

2.5 单克隆筛选：无限稀释到每孔1个细胞的数量，每株细胞铺至少2个96孔板。

细胞数量足够后，验证内源活性并送测。

2.6 获得突变型：如需纯合子，则可能需要重复步骤3-5。

Takara Bio USA, Inc. 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: *******************************United States/Canada Asia Pacific Europe Japan Page 1 of 9Takara Bio USA, Inc. Guide-it™ Recombinant Cas9 (3 µg/µl) User ManualCat. Nos. 632640, 632641 (021121)Table of ContentsI. Introduction (3)A. Summary (3)II. List of Components (3)III. Additional Materials Required (3)A. Electroporation Supplies (3)B. Mammalian Cell Culture Supplies (4)C. General Supplies (4)D. sgRNA Development and Production (4)E. Detection and Characterization of Gene Editing (4)IV. Protocol Overview (5)V. Electroporation Protocol for Neon Transfection System (5)A. Protocol: Preparation of Cells and Media (5)B. Protocol: Preparation of Cas9-sgRNA RNP Complex (6)C. Protocol: Electroporation (6)VI. Electroporation Protocol for 4D-Nucleofector System (7)A. Protocol: Preparation of Cells (7)B. Protocol: Preparation of Cas9-sgRNA RNP Complex (8)C. Protocol: Electroporation (8)VII. References (9)Table of FiguresFigure 1. Protocol overview for Guide-it Recombinant Cas9 (3 µg/µl). (5)I. IntroductionA. SummaryThe CRISPR/Cas9 system has emerged as a powerful tool for gene editing because of its high targetingspecificity, editing efficiency, and ease of use in virtually any organism. CRISPR/Cas9 technologyconsists of two key components that form a complex: Cas9 endonuclease and a single guide RNA(sgRNA) that directs Cas9 to cleave genomic DNA in a sequence-specific manner (Jinek et al. 2012).This RNA-programmable method exploits the error-prone nature of the non-homologous end joiningDNA repair pathway (NHEJ) to generate gene knockouts (via insertion/deletion). The method can also beused to generate knockins via the homology-directed repair (HDR) pathway.CRISPR/Cas9 system components have been delivered successfully into target cells through a variety ofapproaches, including vector-based expression systems, transfection of RNA, and more recently,introduction of Cas9-sgRNA ribonucleoprotein (RNP) complexes. Delivery of Cas9-sgRNA RNPsprovides a fast turnaround for gene-editing experiments while minimizing the likelihood of off-targeteffects compared to vector-based approaches (Sander and Joung 2014), and this approach has beenoptimized for various cell types using microinjection, electroporation, and lipid-mediated transfection(Liang et al. 2015).Guide-it Recombinant Cas9 (3 µg/µl) is a recombinant wild-type Streptococcus pyogenes Cas9 nucleaseexpressed with a C-terminal nuclear-localization signal (NLS)and purified from E. coli for use inCRISPR/Cas9-mediated gene editing experiments. The Cas9 protein solution has been verified to besterile and well-tolerated by mammalian cells when electroporated as a ribonucleoprotein complex (RNP)with a single guide RNA (sgRNA) for knockout experiments, or as an RNP with a donor repair templatefor knockin experiments.II. List of ComponentsGuide-it Recombinant Cas9 (3 µg/µl) (Cat. No. 632641)•100 µg Guide-it Recombinant Cas9 (3 µg/µl)Guide-it Recombinant Cas9 (3 µg/µl) (Cat. No. 632640)• 3 x 100 µg Guide-it Recombinant Cas9 (3 µg/µl)•Store Guide-it Recombinant Cas9 (3 µg/µl) at –70°C.•Avoid repeated freeze/thaw cycles. We recommend preparing aliquots upon initial thawing of Guide-it Recombinant Cas9 (3 µg/µl).III. Additional Materials RequiredThe following reagents/materials are required but not included.A. Electroporation SuppliesUse of this product requires an electroporator, electroporation chamber (typically cuvettes or tips), and anelectroporation buffer that is suitable for your target cells. Here we provide separate guidelines for theNeon Transfection System (Thermo Fisher Scientific, Cat. No. MPK5000) and the 4D-NucleofectorSystem (Lonza, Cat. No. AAF-1002B).B. Mammalian Cell Culture Supplies•Culture medium, supplies, and additives specific to your target cells•Cell culture plates•PBS without Ca2+ or Mg2+•Trypsin/EDTA or equivalent•Humidified incubator (set at 37º C, 5% CO2)C. General Supplies•Single-channel pipettes•Nuclease-free thin-wall PCR tubes or stripsD. sgRNA Development and ProductionCRISPR/Cas9 gene editing requires a custom sgRNA with a user-designed targeting sequence that ishomologous to the target gene or genomic region of interest. Selecting an appropriate DNA sequence at the target region is critical for maximizing the potential for efficient cleavage at the target site and forminimizing the likelihood of non-specific cleavage events. There are several freely available online tools that can be helpful for determining suitable sgRNA target sequences for a given organism and genomic target. For a list of these tools, please refer to:/US/Products/Genome_Editing/CRISPR_Cas9/Resources/Online_tools_for_gui de_RNA_design.NOTE: For many applications, it is advisable to design and test several variant sgRNAs against the same genomic target region.Candidate sgRNAs must ultimately be produced in sufficient quantity for the generation of functionalCas9-sgRNA RNPs. For development and production of user-designed sgRNAs, we recommend either of the following kits:•For constructing and purifying sgRNAs: Guide-it sgRNA In Vitro Transcription Kit (Takara Bio, Cat.No. 632635).•For constructing and purifying sgRNAs, and testing target cleavage efficiencies in vitro: Guide-it Complete sgRNA Screening System (Takara Bio, Cat. No. 632636).E. Detection and Characterization of Gene EditingThese items are recommended for determining the efficiency of gene editing and the nature of the edits:Cat. No. Product Size631443 Guide-it Mutation Detection Kit 100 rxns631448 Guide-it Mutation Detection Kit 25 rxns632611 Guide-it Genotype Confirmation Kit 100 rxns631444 Guide-it Indel Identification Kit 10 rxnsIV. Protocol OverviewPlease read each relevant protocol completely before starting. Successful results depend on understanding and performing the following steps correctly.Figure 1. Protocol overview for Guide-it Recombinant Cas9 (3 µg/µl).V. Electroporation Protocol for Neon Transfection SystemHere we provide protocols for performing knockout and knockin experiments in hiPS cells and CD34-positive stem cells using the Neon Transfection System. While these protocols may serve as a helpful starting point forelectroporation of other cell types as well, further optimization will be required. Please refer to the Neon Transfection System User Manual and manufacturer’s website for detailed operating instructions for the Neon Transfection System.A. Protocol: Preparation of Cells and MediaCultured target cells are harvested, washed, and resuspended in the appropriate buffer.1.Prepare a sufficient number of fresh cells for your experiment.NOTE: Each electroporation requires 1 x 105 cells. However, due to the potential variation of pipetteand tip volumes, we recommend preparing 1.5X the necessary volume of cell suspension (i.e., 1.5 x 105cells) for electroporation with a 10-µl Neon Tip to ensure that there is sufficient volume.2.For hiPS cells (adherent cells), continue to Step3. For CD34-positive stem cells (suspension cells),skip to Step 5.3.Aspirate the medium, wash the cell layer once with PBS (without Ca2+ and Mg2+), and dissociate thecells using TrypLE Select Enzyme (1X) (Thermo Fisher Scientific, Cat. No. 12563011).4.Harvest the cells in growth medium.5.Take an aliquot of the cell suspension and measure the cell density using your preferred method.6.Harvest the cells by centrifugation at 400g for 5 min in a 15-ml conical tube.7.Wash the cells once with PBS (without Ca2+ and Mg2+), and then resuspend hiPS cells in Buffer R andCD34-positive stem cells in Buffer T (included with Neon kits) at a concentration of 2 x 107 cells/ml(i.e., 1.5 x 105 cells in 7.5 µl).NOTE: Use Resuspension Buffer R for established adherent and suspension cells as well as primaryadherent cells, and use Resuspension Buffer T for primary blood-derived suspension cells.8.Keep the cell suspension on ice until use.B. Protocol: Preparation of Cas9-sgRNA RNP ComplexCas9 and sgRNA components are combined to form RNP complexes for electroporation.1.Thaw Guide-it Recombinant Cas9 (3 µg/µl) and sgRNA solutions at room temperature.NOTE: We recommend preparing aliquots upon initial thawing of Guide-it Recombinant Cas9(3 µg/µl) to avoid repeated freeze/thaw cycles.bine the following components in a 200-µl PCR tube to mix the Cas9 protein and sgRNA at a 5:1mass ratio. The molar ratio of Cas9 protein to sgRNA will be approximately 1:1 in this mixture, andthe total volume will be 7.5 µl. Be sure to use the same buffer that was used to resuspend the cells.NOTE: The reaction volume indicated below is 1.5X the required volume.Per reaction:0.45 μl* sgRNA (e.g., 1 µg/µl)0.75 μl Guide-it Recombinant Cas9 (3 µg/µl)6.3 μl* Resuspension Buffer R or T7.5 μl Total volume*The added volume of sgRNA will vary depending on sgRNA concentration, and the added volume ofResuspension Buffer should be adjusted such that the total reaction volume is 7.5 µl. The volumesindicated above are based on a sgRNA concentration of 1 µg/µl.NOTE: Make a master mix if you are performing multiple electroporations.NOTE: The optimal amount of RNP complex may vary for different cell types.NOTE: To maximize electroporation efficiency, the combined volume of the Cas9 and sgRNA solutionsshould be ≤20% of the total volume of the Cas9-sgRNA RNP complex reaction (e.g., for the 7.5-µlreaction specified above, the combined volume of the sgRNA and Cas9 solutions should be ≤1.5 µl).NOTE: If you plan to use donor DNA to induce HDR-mediated knockin, add the DNA after thesubsequent incubation step (Step 3). We recommend using ≤1 µg of DNA for knockin experiments.Adjust the volume of Resuspension Buffer R or T included in the reaction such that the final volumeupon addition of donor DNA is 7.5 µl.3.Mix the reaction well by gently pipetting up and down. Incubate using a thermal cycler preheated to37°C with the following program:37°C 5 min4°C hold4.OPTIONAL: Add donor DNA and keep on ice until use.C. Protocol: ElectroporationCas9-sgRNA RNPs are electroporated into target cells.1.Fill the Neon Tube with 3 ml of Buffer E (included with Neon kits) and insert the Neon Tube into theNeon Pipette Station.ing the touchscreen on the Neon system, set up the electroporation parameters as follows:Pulse voltage / Pulse width / Pulse number = 1100 v / 20 ms / 2 pulsesNOTE: We have used these parameters successfully for hiPS cells and CD34-positive stem cellsusing the Neon Transfection System. Optimization of electroporation parameters will be required fordifferent target cell types. Suggested parameters for different cell types are included in thesupplementary material for (Liang et al. 2015).3.Gently resuspend the cells by tapping, and transfer 7.5 µl of the cell suspension into the PCR tubecontaining the 7.5 µl of Cas9-sgRNA RNP complex solution.4.Mix well by gently pipetting up and down.5.Insert the Neon Pipette into the Neon Tip and confirm that the pipette and tip are tightly connected.ing the Neon Pipette, aspirate the mixture slowly into the Neon Tip.NOTE: Avoid any air bubbles in the tip. If you notice air bubbles, place the sample back into thePCR tube and aspirate again into the tip without any air bubbles.7.Insert the Neon Pipette into the Neon Tube placed in the Neon Pipette Station and run the program.8.Remove the pipette very carefully and transfer the cells into a cell culture plate with pre-warmedmedium.NOTE: Use an appropriate well plate for your target-cell type. We had success using 24 and 48-wellplates for CD34-positive stem cells and hiPS cells, respectively. hiPS cells typically require greaterconfluence than regular adherent cells.9.Shake the plate appropriately to disperse the cells and incubate at 37°C in a humidified incubator with5% CO2 until the next necessary procedure.VI. Electroporation Protocol for 4D-Nucleofector SystemHere we provide protocols for performing knockout and knockin experiments in Jurkat and CD34-positive stem cells using the 4D-Nucleofector System with 16-well Nucleocuvette Strips. While these protocols may serve as a helpful starting point for electroporation of other cell types as well, further optimization will be required. Please refer to the 4D-Nucleofector System User Manual and manufacturer’s website for more detailed information.A. Protocol: Preparation of CellsCultured target cells are harvested, washed, and resuspended in the appropriate solution.1.Prepare a sufficient number of fresh cells for your experiment.NOTE: Each electroporation requires 2 x 105 cells.2.Take an aliquot of the cell suspension and measure the cell density using your preferred method.3.Harvest the cells by centrifugation at 400g for 5 min in a 15-ml conical tube.4.Wash once with PBS (without Ca2+ and Mg2+), and then resuspend Jurkat cells in SE NucleofectorSolution (with supplement) and CD34-positive stem cells in P3 Nucleofector Solution (withsupplement) at a concentration of 1 x 107 cells/ml (i.e., 2 x 105 cells in 20 µl).NOTE: Please refer to the 4D-Nucleofector System User Manual and manufacturer’s website formore information about working with other cell types.NOTE: 20 µl of cell suspension will be needed per well of the Nucleocuvette Strip.5.Keep the cell suspension on ice until use.B. Protocol: Preparation of Cas9-sgRNA RNP ComplexCas9 and sgRNA components are combined to form RNP complexes for electroporation.1.Thaw Guide-it Recombinant Cas9 (3 µg/µl) and sgRNA solutions at room temperature.NOTE: We recommend preparing aliquots upon initial thawing of Guide-it Recombinant Cas9(3 µg/µl) to avoid repeated freeze/thaw cycles.bine the following components in a 200-µl PCR tube to mix the Cas9 protein and sgRNA at a 5:1mass ratio:Per reaction (e.g. 5 µl):2μl* sgRNA (e.g., 1 µg/µl)3.3 μl Guide-it Recombinant Cas9 (3 µg/µl)5.3 μl Total volume*The added volume of sgRNA will vary depending on sgRNA concentration. The volume indicated aboveis based on an sgRNA concentration of 1 µg/µl.NOTE: Make a master mix if you are performing multiple electroporations.NOTE: The RNP volume required for efficient transfection needs to be optimized for different celltypes. Usually ≤10 µl of the RNP solution will be tested for electroporation in each well of the 16-well Nucleocuvette Strip.3.Mix the reaction well by gently pipetting up and down. Incubate using a thermal cycler preheated to37°C with the following program:37°C 5 min4°C HoldC. Protocol: ElectroporationCas9-sgRNA RNPs are electroporated into target cells.bel wells of Nucleocuvette Strips to be used for electroporation.bine 20 µl of cell suspension with 5 µl Cas9-sgRNA RNP complex solution in each well of theNucleocuvette Strip, and mix well by gently pipetting up and down.NOTE: If you plan to use donor DNA to induce HDR-mediated knockin, add the donor DNA at thisstep.3.Select the program CL-120 for Jurkat cells or the program D0-100 for CD34-positive stem cells.4.Insert the Nucleocuvette Strip into the Nucleofector machine and run the program.5.Add 80 µl of pre-warmed medium to each cuvette and allow electroporated cells to recover for 12min post-transfection at room temperature.6.Gently collect the cells along with the media from each well and transfer to individual wells of a 48-well plate containing pre-warmed medium.7.Shake the plate appropriately to disperse the cells and incubate at 37°C in a humidified incubator with5% CO2 until the next necessary procedure.VII. References1.Jinek, M., Chylinsky, K., Fonfara, I., Hauer, M., Doudna, J.A., & Charpentier, E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337(6096), 816–21 (2012).2.Liang, X. et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J.Biotechnol.208, 44–53 (2015).3.Sander, J.D. & Joung, J.K. CRISPR-Cas9 systems for genomic editing, regulation and targeting. Nat.Biotechnol. 32, 347-55 (2014).Contact UsCustomer Service/Ordering Technical Supporttel: 800.662.2566 (toll-free) tel: 800.662.2566 (toll-free)fax: 800.424.1350 (toll-free) fax: 800.424.1350 (toll-free)web: web: e-mail: **********************e-mail: ********************Notice to PurchaserOur products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.Your use of this product is also subject to compliance with any applicable licensing requirements described on the product’s web page at . It is your responsibility to review, understand and adhere to any restrictions imposed by such statements© 2021 Takara Bio Inc. All Rights Reserved.All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at .This document has been reviewed and approved by the Quality Department.。

cas9中scaffold template -回复Cas9蛋白酶是CRISPR-Cas9系统的核心组成部分之一，它在基因组编辑中起着至关重要的作用。

Cas9蛋白酶是通过依赖于一种称为scaffold的RNA分子来实现与目标DNA序列特异性结合的。

本文将详细介绍Cas9中scaffold模板的重要性以及其在基因编辑中的作用。

首先，让我们了解一下CRISPR-Cas9系统。

CRISPR是一种反映了细菌和古菌天然免疫系统的新技术，可以实现高效、精确和经济的基因组编辑。

CRISPR系统的核心组分包括Cas9蛋白酶和两种小RNA分子：crRNA 和tracrRNA。

Cas9蛋白酶通过与合适的sgRNA（single guide RNA）结合，指导其靶向特定的基因序列。

sgRNA是由crRNA和tracrRNA两种小RNA分子合成的单个RNA分子。

在整个过程中，scaffold模板也扮演着重要的角色。

Cas9中的scaffold模板在CRISPR-Cas9系统中的作用是什么？scaffold 模板对于Cas9蛋白酶与目标DNA的结合起着至关重要的作用。

一方面，scaffold模板可能通过提供基于互补链的RNA序列来与目标DNA序列匹配，从而引导Cas9蛋白酶特异性地识别和结合到目标位点。

另一方面，scaffold模板还可能提供其他结构上启动和稳定RNA结合Cas9蛋白酶所需的要素。

总之，scaffold模板是确保Cas9蛋白酶与目标DNA的精确结合的关键因素。

为了更好地理解Cas9中scaffold模板的功能，我们需要了解其结构。

scaffold模板通常由互补链的RNA序列组成，其中还包括其他一些结构模块，如戈登结构、非标准碱基对结构等。

这些结构模块有助于增加Cas9与目标DNA序列的特异性结合和识别，从而提高编辑效率和准确性。

现在让我们详细介绍一下Cas9中scaffold模板在基因组编辑中的应用。

在CRISPR-Cas9系统中，通过将特定的sgRNA合成导入到目标细胞中，Cas9蛋白酶会被激活，并与sgRNA结合形成有效的编辑复合物。

【评测】Alt-RS.p.Cas9-GFP荧光标签cas9核酸酶Cas9-GFP（或 RFP）核酸酶为您的CRISPR基因编辑研究增添⾊彩#IDT新款荧光标签cas9核酸酶Alt-R S.p.Cas9-GFP 和 Alt-R S.p.Cas9-RFP，便于转染后可视化或FACS分选从表达核酸酶和 eGFP 或 RFP 的⼤肠杆菌菌株中纯化的重组化脓性链球菌 Cas9 核酸酶。

包含核定位序列 (NLS) 和 C 端 6-His 标签。

以 10 µg/µL 的溶液形式提供。

100 µg 荧光融合 Cas9 核酸酶 = 530 pmol。

北京泽平供应各类CRISPR 核酸酶系列，请搜索“泽平科技”进⼊咨询报价。

相关引⽤⽂献：Paix A、Rasoloson D、Folkmann A、Seydoux G. （2019）使⽤双链 DNA 供体通过基因组⼯程快速标记⼈类蛋⽩质与荧光报告基因。

Curr Protoc Mol Biol。

doi：10.1002/cpmb.102Vakulskas C、Dever D 等。

(2018) 作为核糖核蛋⽩复合物交付的⾼保真 Cas9 突变体能够在⼈类造⾎⼲细胞和祖细胞中进⾏有效的基因编辑。

⾃然医学。

doi：10.1038/s41591-018-0137-0该报告描述了 Cas9 变体的分离，该变体在以 RNP 格式交付时显⽰出卓越的⽬标脱靶率。

在难以编辑的原代细胞中的治疗相关基因座上实现了强⼤的靶向编辑，⽽整体脱靶编辑显着减少。

研究中使⽤的⾼保真 Cas9 酶现在可以从 IDT 以Alt-R HiFi Cas9 核酸酶 V3 的形式从市场上买到。

Tröder SE、Eber LK 等。

(2018) ⼀种优化的电穿孔⽅法，⽤于在⼩⿏受精卵中进⾏有效的 CRISPR/Cas9 基因组编辑。

PLoS ⼀， 13 （5）： e0196891。

基因治疗中的基因敲除筛选与验证方法基因治疗是近年来快速发展的一项前沿技术，它通过修复或替代异常基因以治疗遗传性疾病和其他疾病。

基因治疗中的一个重要步骤是基因敲除筛选与验证方法，它可以帮助研究人员确定目标基因，并确保敲除的准确性和有效性。

本文将讨论基因治疗中常用的基因敲除筛选与验证方法。

一、CRISPR/Cas9系统CRISPR/Cas9系统是目前最常用的基因敲除方法之一。

该系统利用一种特殊的RNA分子（CRISPR RNA），与一种酶（Cas9）结合，形成一种RNA-蛋白复合体。

这个复合体通过与目标基因的DNA序列匹配，以导致双链断裂。

当细胞试图修复这个断裂时，往往会引起基因突变或插入/缺失等错误修复，最终导致目标基因的敲除。

CRISPR/Cas9系统具有准确、高效和简便的优点，因此被广泛应用于基因治疗中的基因敲除。

研究人员可以通过合成适当的CRISPR RNA和Cas9蛋白，将其导入细胞，并使用基因编辑技术检测目标基因是否被成功敲除。

二、RNA干扰（RNAi）技术RNA干扰技术是另一种常用的基因敲除筛选方法。

该技术利用小干扰RNA （siRNA）或小发夹RNA（shRNA），通过与目标基因的mRNA相结合，引导RNA酶复合物切割目标mRNA，从而降低或抑制该基因表达。

使用RNA干扰技术进行基因敲除筛选具有一定的优势。

首先，该技术广泛适用于多种细胞类型和体外和体内实验。

其次，通过设计合适的siRNA或shRNA，可以选择靶向特定基因的序列，以实现高度特异性的基因敲除。

三、逆转录聚合酶链反应（RT-PCR）逆转录聚合酶链反应（RT-PCR）是一种常用的基因敲除验证方法。

该方法通过首先将mRNA转录为互补DNA（cDNA），然后使用特定引物进行PCR扩增。

最终，扩增产物可以通过凝胶电泳和测序技术来确定是否成功敲除了目标基因。

RT-PCR在基因敲除验证中的作用非常重要。

通过检测cDNA的存在与否，研究人员可以确定目标基因是否成功敲除，并进一步评估其在细胞和组织中的表达水平。

ptyne载体验证cas9实验步骤《ptyne载体验证cas9实验步骤》引言：Cas9蛋白是一种CRISPR/Cas系统中的核酸酶，可以与RNA相结合，从而实现基因编辑、转录调控和基因组追踪等功能。

为了实现高效的基因编辑，研究人员通常需要运用载体来将Cas9蛋白导入到目标细胞中，并进行验证。

本文将详细介绍如何使用ptyne载体验证Cas9蛋白的实验步骤。

步骤一：准备实验所需材料在进行Cas9验证实验之前，我们需要准备以下实验材料：- ptyne载体：该载体通常用于将Cas9蛋白导入到目标细胞中。

它包含了所需的启动子、限制酶切位点以及其他必要的元件。

可以通过购买或者自行构建来获取。

- Cas9蛋白：可以购买纯化的Cas9蛋白，也可以通过表达系统自行获得。

- 目标细胞：根据实验需要，选择适当的细胞系进行验证。

常用的目标细胞有HEK293细胞、Hela细胞等。

- 目标基因：根据实验需要选择一个特定的基因进行验证。

步骤二：将Cas9基因导入到ptyne载体中1. 获得Cas9编码序列：将所需的Cas9基因序列通过PCR扩增或从已有的载体中提取出来。

2. 制备ptyne载体：将ptyne载体线性化并准备好限制酶切位点。

3. 进行酶切反应：将Cas9基因和ptyne载体分别与适当的限制酶一起进行酶切反应。

这样可以在载体中创建一个合适的切口，使Cas9基因能够被正确插入载体。

4. 进行连接反应：将酶切后的Cas9基因和ptyne载体通过连接酶进行连接。

这样可以将Cas9基因正确地导入到ptyne载体中。

步骤三：将ptyne载体与目标细胞转染1. 制备转染系统：根据实验需要选择适当的转染试剂，并按照厂商提供的操作指南制备不同浓度的转染试剂。

2. 细胞培养与维护：将目标细胞在培养皿中培养至适当的密度，并保证其健康状态。

3. 转染操作：将制备好的ptyne载体与转染试剂按照适当比例混合，形成转染复合物。

将复合物加入到培养皿中并进行适当时间的培养。

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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!1. 设计 sgRNA（单向导 RNA）：选择要敲除的目标基因，并确定其外显子和内含子的位置。

ptyne载体验证cas9实验步骤-回复【ptyne载体验证cas9实验步骤】引言：CRISPR-Cas9系统是近年来被广泛应用于基因工程和生物医学研究中的一种重要技术。

其中Cas9是CRISPR-Cas9系统中的核酸酶，在定点剪接DNA中发挥关键作用。

为了有效利用Cas9进行基因编辑，研究人员通常需要验证所选择的载体是否能够稳定表达Cas9。

本文将详细介绍ptyne 载体验证Cas9实验的步骤及注意事项。

一、实验准备1. 购买所需试剂和材料，包括ptyne载体、Cas9编码序列、质粒提取试剂盒等。

2. 制备所需培养基，如LB培养基或甘露醇培养基。

3. 提前培养所需的细菌菌种，如大肠杆菌。

4. 准备好所需的培养器具和设备，如离心管、离心机、恒温培养器等。

二、构建质粒1. 将ptyne载体和Cas9编码序列经酶切，并通过凝胶电泳分离线性质粒和酶切产物。

2. 将线性质粒回收，并利用质粒提取试剂盒提取纯化质粒。

三、转化细菌1. 将质粒导入目标宿主细菌中，如大肠杆菌。

可以通过热激转化或电转化等方法进行转化。

2. 将转化后的细菌涂布于含有抗生素的培养基上，筛选可以稳定表达Cas9的阳性菌落。

四、鉴定阳性菌落1. 从培养基上挑选出阳性菌落，并进行扩增培养。

2. 利用PCR方法检测扩增物是否正确。

五、质粒提取1. 利用质粒提取试剂盒提取阳性菌落中的质粒。

2. 利用凝胶电泳鉴定提取到的质粒是否纯化。

六、质粒测序1. 将提取到的质粒送去测序，以确保Cas9编码序列正确无误。

七、表达验证1. 利用免疫印迹或荧光刺激等方法验证Cas9是否能够稳定表达。

2. 进一步验证Cas9的功能性，如通过利用CRISPR-Cas9系统进行基因敲除或基因编辑等实验。

注意事项：1. 在所有步骤中，实验室必须遵循基本的操作规范，如佩戴实验手套、操作在无菌条件下进行等。

2. 选择合适的培养条件和培养时间以确保细菌的正常生长和繁殖。

3. 注意使用适当的抗生素浓度筛选阳性菌落，避免抗性菌株的出现。

一、材料试剂准备1）质粒：pSpCas9(BB)-2A-GFP (Addgene plasmid ID: 48138)，用于转染cas9，该质粒含有Cas9与GFP，Cas9的nickase活性将用于特定目的基因的ko，GFP可做为转染标签。

pSpCas9(BB) (Addgene plasmid ID: 42230)，若实验用sgRNA为PCR扩增纯化产物，则需要该质粒做为U6的模版。

pUC19(Invitrogen, cat.no.15364-011)可用于构建sgRNA，若用PCR产物进行转染，则需要该质粒来进行共转染，做为DNA carrier。

上述三种质粒，根据实验选择sgRNA的表达方式（PCR产物/ 单载体系统/ 双载体系统）来确定具体使用哪种。

2）超纯水，DNase/RNase-free (Life Technologies, cat. no. 10977-023)3）高保真聚合酶，Kapa HiFi (Kapa Biosystems), PfuUltra (Agilen)，Herculase II fusion polymerase 均可，只需保真效果好，扩增过程不产生突变。

4）Taq DNA polymerase with standard Taq buffer (NEB, cat. no. M0273S)用于一般检测。

5）QIAquick gel extraction kit (Qiagen, cat. no. 28704)6）QIAprep spin miniprep kit (Qiagen, cat. no. 27106)7）Fast Digest BbsI (BpiI) (Fermentas/Thermo Scientific, cat. no. FD1014)，如需要将sgRNA构建到pSpCas9(BB)-2A-GFP质粒上，则需要该酶。

8）T7 DNA ligase with 2× rapid ligation buffer (Enzymatics, cat. no. L602L). 或者T4 DNA ligase，二者无区别。