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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基因敲除原理
CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats）是最新出现的一种由RNA指导Cas核酸酶对靶向基因进行特定DNA修饰的技术。
CRISPR 是细菌和古细菌为应对病毒和质粒不断攻击而演化来的获得性免疫防御机制。
在这一系统中,crRNA（CRISPR-derived RNA)通过碱基配对与tracrRNA(trans-activating RNA）结合形成双链RNA，此tracrRNA/crRNA二元复合体指导Cas9蛋白在crRNA引导序列靶定位点剪切双链DNA达到对基因组DNA进行修饰的目的。
Cas9结合gRNA，gRNA 的长度约为80个核苷酸,包含两个区域:gRNA 5' 端前20个核苷酸对应于靶标DNA，能结合在靶DNA 上的约60个核苷酸（gRNA 长度取决于表达gRNA 的质粒）形成一个发夹结构，这个结构能帮助gRNA 与Cas9结合,并由此指导与DNA 的结合.
通过gRNA上的靶点序列，在目标基因组上找到靶点序列，并揭开双螺旋，Cas9将剪切DNA双链，造成DNA双链断裂。
Cas9使用简单，可满足多个靶点同时操作。
Insertion /deletion NHEJ HDR
gRNA
Cas9
Donor vector
基因敲除小鼠流程：。
1CRISPR-Cas系统的研究进展CRISPR(clustered regularly interspaced short palindromic repeats)，即串联的、间隔的短回文重复序列，最早在1987年研究大肠杆菌的碱性磷酸酶基因时被发现[1]。
随后在细菌和古细菌的基因组中也发现大量存在CRISPR，研究证实它能够保护自身抵御外来病毒和质粒的入侵[2]，作用机制是依靠crRNA(CRISPR RNA)和tracrRNA(trans-activating crRNA)结合并引导Cas蛋白对外源DNA进行特异性降解[3]。
已发现的CRISPR-Cas系统有三种类型：Ⅰ型，Ⅱ型和Ⅲ型，其中以Ⅱ型最为简单，只需一种Cas蛋白，即通过RNA 介导核心蛋白Cas9识别并切割靶序列，引起DNA双链断裂[2]。
受自然界中CRISPR-Cas系统的启发，主要对来自于化脓性链球菌(Streptococcus pyogenes)的Ⅱ型CRISPR-Cas系统进行人为改造和利用，目前已经将其发展成为一种新型的基因编辑技术，实现基因敲除、插入、定点突变和组合编辑[4]，并成功应用于大肠杆菌、酿酒酵母、家蚕、果蝇和人类细胞等[5]。
和传统的基因编辑技术相比，这一新技术具有成本低、操作简便、效率高的优点[6]。
2 CRISPR-Cas系统的组成与机制典型的Ⅱ型CRISPR-Cas系统基因座包含tracrRNA基因、Cas蛋白编码基因(cas9、cas1、cas2和csn2)、CRISPR基因座（引导序列、间隔序列和重复序列）这三个部分[6]。
Ⅱ型CRISPR-Cas系统的作用机制可分为三个阶段，第一是高度可变间隔序列的获得（图1），第二是CRISPR-Cas系统基因座的表达，第三是对外源遗传物质的降解[6]（图2）。
Cas1、Cas2和Csn2蛋白与新间隔序列的获得相关。
与间隔序列同源的外源遗传物质上的原间隔序列(protospacer)，其下游存在一段保守序列，被称为PAM(protospacer adjacent motifs)[7]。
使用CRISPR-CAS系统构建可遗传的基因敲除小鼠和大鼠致编辑：CRISPR-CAS系统已经成为一种细胞和模式生物中有效的基因编辑技术。
我们使用CRISPR-CAS系统，通过同时注入两种单导向RNA靶向Uhrf2,同时带入Cas9 mRNA来诱导小鼠的DNA片段缺失。
此外，我们通过一种单一的显微注射方法得到了敲除Mc3R和Mc4R两种基因的大鼠。
在小鼠和大鼠中均观察到较高的种系转移效率（突变可遗传）。
成簇有序间隔短回文重复关联蛋白系统（CRISPR-CAS系统）是一种在细菌和古细菌中演变的针对病毒和质粒入侵的基于RNA的后天免疫系统。
【Bamboo注：该系统由一段Cas基因（双链DNA核酸酶）加一段特异序列组成，Cas作用为结合导向RNA,切割目的基因，导向RNA为CRISPR序列转录而成，有二级结构】根据作用机制不同，CRISPR-CAS系统目前有三种类型。
在第二类型（下文称该系统）中，CRISPR序列转录RNA（crRNA）和反式激活RNA(TraceRNA)结合后有能力引导Cas9核酸内切酶到特定的序列，从而导致目标DNA双链缺失。
【Bamboo注：机理：摄取了病毒DNA后，CRISPR 序列在转录产物tracrRNA,表达产物CAS蛋白，RNA酶共同作用下产生导向RNA,导向RNA含有病毒DNA序列，可遗传给下一代，再遇到病毒DNA时将其剪切】之前的研究表明在哺乳动物中有多种基因工程使用RNA介导的Cas9核酸酶系统。
最近，使用该系统进行高效基因编辑已经在斑马鱼、小鼠和细菌中实现。
几个小组也证明通过该系统介导的在细胞和斑马鱼中基因靶向效率与ZFNs（锌指核酸酶）和TALENs（转录活化因子效应核酸酶）【Bamboo注：另外两种常见DNA编辑方式】相似或较高。
虽然已经有报道在单个小鼠胚胎中可使用该系统打乱多个基因，但是在动物体中尚未见该系统介导的突变种系转移。
此外，长的特异的基因组DNA片段能否被该系统敲除也是未知的。
CRISPR-Cas9文库技术原理及应用CRISPR-Cas9技术原理CRISPR-Cas9技术凭借着成本低廉，操作方便，效率高等优点，CRISPR-Cas9技术迅速风靡全球的实验室，成为了生物科研的有力帮手，是继“锌指核酸内切酶（ZFN）”、“类转录激活因子效应物核酸酶（TALEN）”之后出现的第三代“基因组定点编辑技术”。
CRISPR-Cas9系统最初在大肠杆菌基因组中被发现，是细菌中抵抗外源病毒的免疫系统。
CRISPR-Cas9系统由两部分组成，一部分是用来识别靶基因组的，长度为20bp左右的sgRNA 序列，另外一部分是存在于CRISPR位点附近的双链DNA核酸酶——Cas9，能在sgRNA的引导下对靶位点进行切割，最终通过细胞内的非同源性末端连接机制（NHEJ）和同源重组修复机制（HDR）对形成断裂的DNA进行修复，从而形成基因的敲除和插入，最终实现基因的（定向）编辑。
与前两代技术相比，CRISPR-Cas9技术最大的突破是不仅可以对单个基因进行编辑，更重要的是可以同时对多个基因进行编辑，这也为全基因组筛选提供了有效的方法。
目前比较常见的文库类型包括：●CRISPR-Cas9 knock out文库●CRISPR panal文库●CRISPRa/i文库●psgRNA文库CRISPR-Cas9文库建库流程●靶位点确认及sgRNA文库设计●sgRNA文库芯片合成●sgRNA文库构建●QC验证文库质量●sgRNA文库慢病毒包装●感染稳定细胞株●药物筛选实验●细胞表型筛选●NGS测序验证功能基因CRISPR-Cas9文库应用方向1、药物靶点确定与验证CRISPR-Cas9筛选技术可以应用于药物靶点筛选中，通过大规模筛选技术，可以系统的分析、验证一些与抗药性相关的基因，从而为疾病治疗提供相关数据。
SCIENCE发表文章[1]，研究人员利用CRISPR-Cas9文库筛选人类黑色素瘤A375细胞中的18,080个基因进行筛选，最终发现NF2、CUL3等4个基因参与了黑色素瘤A375细胞中的耐药调节过程。
1CRISPR-Cas系统的研究进展CRISPR(clustered regularly interspaced short palindromic repeats)，即串联的、间隔的短回文重复序列，最早在1987年研究大肠杆菌的碱性磷酸酶基因时被发现[1]。
随后在细菌和古细菌的基因组中也发现大量存在CRISPR，研究证实它能够保护自身抵御外来病毒和质粒的入侵[2]，作用机制是依靠crRNA(CRISPR RNA)和tracrRNA(trans-activating crRNA)结合并引导Cas蛋白对外源DNA进行特异性降解[3]。
已发现的CRISPR-Cas系统有三种类型：Ⅰ型，Ⅱ型和Ⅲ型，其中以Ⅱ型最为简单，只需一种Cas蛋白，即通过RNA 介导核心蛋白Cas9识别并切割靶序列，引起DNA双链断裂[2]。
受自然界中CRISPR-Cas系统的启发，主要对来自于化脓性链球菌(Streptococcus pyogenes)的Ⅱ型CRISPR-Cas系统进行人为改造和利用，目前已经将其发展成为一种新型的基因编辑技术，实现基因敲除、插入、定点突变和组合编辑[4]，并成功应用于大肠杆菌、酿酒酵母、家蚕、果蝇和人类细胞等[5]。
和传统的基因编辑技术相比，这一新技术具有成本低、操作简便、效率高的优点[6]。
2 CRISPR-Cas系统的组成与机制典型的Ⅱ型CRISPR-Cas系统基因座包含tracrRNA基因、Cas蛋白编码基因(cas9、cas1、cas2和csn2)、CRISPR基因座（引导序列、间隔序列和重复序列）这三个部分[6]。
Ⅱ型CRISPR-Cas系统的作用机制可分为三个阶段，第一是高度可变间隔序列的获得（图1），第二是CRISPR-Cas系统基因座的表达，第三是对外源遗传物质的降解[6]（图2）。
Cas1、Cas2和Csn2蛋白与新间隔序列的获得相关。
与间隔序列同源的外源遗传物质上的原间隔序列(protospacer)，其下游存在一段保守序列，被称为PAM(protospacer adjacent motifs)[7]。
cas9蛋白结构域摘要：1.CAS9 蛋白的概述2.CAS9 蛋白的结构域3.CAS9 蛋白结构域的功能4.CAS9 蛋白结构域的应用正文：1.CAS9 蛋白的概述CAS9（CRISPR-associated protein 9）是一种核酸内切酶，属于CRISPR-Cas 系统中的核心组成部分。
CRISPR-Cas 系统是一种原核生物天然免疫系统，可以有效地抵御外来DNA，如噬菌体等。
CAS9 蛋白在CRISPR-Cas 系统中扮演着“分子剪刀”的角色，可精确识别并切割外源DNA，从而保护宿主细胞免受入侵。
2.CAS9 蛋白的结构域CAS9 蛋白的结构由多个结构域组成，主要包括以下四个结构域：（1）核酸结合结构域（N-terminal DNA-binding domain，简称BD）：该结构域负责识别和结合目标DNA 序列，从而定位切割位点。
（2）RNA 结合结构域（RNA-binding domain，简称RBD）：该结构域负责与向导RNA（guide RNA）结合，使CAS9 蛋白能够识别并切割特定的目标DNA 序列。
（3）中央结构域（central domain，简称CD）：该结构域在CAS9 蛋白的活性中心，负责形成蛋白质-RNA-DNA 三元复合物，从而实现切割作用。
（4）催化结构域（catalytic domain，简称DD）：该结构域包含切割活性中心，可与中央结构域协同作用，实现对目标DNA 的切割。
3.CAS9 蛋白结构域的功能CAS9 蛋白的各个结构域具有不同的功能，共同协作以完成对目标DNA 的识别和切割。
核酸结合结构域负责识别目标DNA 序列，RNA 结合结构域则确保切割的准确性。
中央结构域使CAS9 蛋白能够与目标DNA 和向导RNA 形成稳定的三元复合物，从而为催化结构域提供合适的空间和环境进行切割。
4.CAS9 蛋白结构域的应用CAS9 蛋白结构域的研究不仅加深了我们对CRISPR-Cas 系统的理解，还为基因编辑技术的发展提供了重要依据。
一文教你用CRISPRCas9在细菌中敲除特定基因一文教你利用CRISPR-Cas9系统快速精准的敲除靶标基因DNA Fragment Knockout Mediated by Dual guide RNAs/Cas9 System in lentivirus backbonePreviously, we described the method for DNA fragment knockout in the genome mediated by two dual guide RNAs. Compared with the single guide RNA mediated gene mutation, the dual guide RNAs/Cas9 system has a great deal of advantages, such as higher efficiencies, easy to screen the positive clones, much more optional for guide RNA design ( you can design two guide RNAs in the intron), selectively knocking out one isoform of genes without affecting other isoforms etc. The primary version of Dual guide RNAs/ Cas9 system is in the common mammalian expression cassettes, which is limited for using for primary and hard transfected cells. Now, to resolve these hurdles, we developed the Dual guide RNAs/ Cas9 system in lentivirus backbone, which contains lentiCRISPR-Dual-Puro, lentiCRISPR-Dual-GFP, Topo-Dual, pdR8.2, pMD2.GWhat is the CRISPR-Cas9 system?Figure 1. Overview of CRISPR-Cas9 systemCRISPR-Cas9 system is a widely used genome editing tool, which derives from the bacteria adaptive immune mechanism. As an engineered version, nowadays CRISPR-Cas9 system contains one enzyme Cas9 and one guide RNA. This guide RNA can direct cas9 to specific genome locus for further digestion. As shown in Figure 1.So far, CRISPR-Cas9 system has two main applications: Gene knock-out and Gene knock-in. For gene knockout, once DNA double strand breaks are induced, the cells will repair the break through NHEJ DNA repair pathway which will cause tiny deletion or insertion. For gene knock in. with the homolog recombination template, the cells will be inserted one specific foreign sequence in genome.Fragment deletion (Gene knockout) mediated by two digesting eventsCompared with previous strategy, this protocol uses a different approach to achieve deletion of DNA fragment in specified size. In this protocol, two guide RNAs targeting adjacent sites of certain region are needed, and two digesting events mediated by Cas9 will cause the degradation of internal fragment. Then the ends will be ligated through NHEJ pathway. In the end, this strategy can cause fragment deletion in specified size. With this protocol, one or some exons can be deleted, or whole genes can be deleted. The diagram is shown in Figure 2.To achieve higher knockout efficiency, vector carrying onecas9 protein and two guide RNA expressing cassettes is generated.Figure 2. Diagram of Cas9-2hitKO systemPlasmids for this protocolTo achieve the higher editing efficiencies and crash the hard transfected cells hurdles, lenti viral vector harboring one cas9 protein and two guide RNA expressing cassettes is generated. In this protocol, three plasmids are involved. lentiCRISPR-Dual-puro and lentiCRISPR-Dual-GFP contain cas9 and one Guide RNA expressing cassette. Adding another MCS is for the inserting of the second Guide RNA expressing cassette. Topo-Dual is generated for the second guide RNA expressing cassette.lentiCRISPR-Dual-GFP contains EGFP for fluorescent sorting; lentiCRISPR-Dual-puro contains puromycin resistant gene for drug selection.Figure 3. Map for lentiCRISPR Dual PuroFigure 4. Map for lentiCRISPR Dual GFPFigure 5. Map for Topo-DualAssembly of Guide RNAAccording to Zhang Feng lab’s protocol, guide RNA can be easily assembled through an ingenious strategy. This strategy is shown in Figure 6. When Guide RNA scaffold is digested by BbsI, an annealed duplex with specific sticking ends (as 20 Nucleotides’ target) will be ligated in this plasmid through T4 DNA ligase.Figure 6. Strategy for guide RNA assemblyProcedureI. Pick Cas9 target with minimal off-targets potentialsWebsite for target prediction/. The brief manual is as below (Figure 7.)1. Input no more than 250 nts’ genomic DNA sequence to the input box, and pick the corresponding species. Then click button ‘submit’.2. Click button ‘Guides & offtargets’.3. Choose targets whose scores > 90. The targets can be in both orientations.Figure 7. Submission of prediction of guide RNA targetII. Pick a pair of Cas9 targets to delete the objective DNA fragmentTo delete a genomic DNA fragment, two targets recognized by guide RNA are indispensable. According to previous test, as long as 1M bps sequence can be deleted efficiently. However, fragment deletion of <10 kbs should be fine and efficient enough. The criteria of choice on target pair is as followed, ( Figure 8 and Figure 9 are two examples)a. If the interested gene is short (about 10 kbs, or a little larger), just delete the whole gene including promoter region.b. If the interested gene is large, deleting the most important exon or exons should be a good choice.c. If the interested gene is large, an alternative is deleting a part of the gene including whole promoter regiond. If the interested gene was knockout by traditional strategy previously, just delete the same region.Figure 8. Cdkn2a Cas9-targetingFigure 9. PTEN Cas9-targetingIII. Construct CRISPR-Cas9 vector carrying two Guide RNA expressing cassettesFigure 10. Overview of construction1. Synthesis target oligos.Oligo-Forward: CACC-(N)20Oligo-Reverse: AAAC-(N)20R CIf the first nucleotide of the target sequence is not ‘G’, just add one ‘G’ to the 5’ end. The oligos becomeForward: CACC-G(N)20Reverse: AAAC-(N)20R C2. AnnealingOn thermal cycler with heat lid.37 ℃, 30 min95 ℃, 5 minRamp down to 25℃at 0.1℃/sec. (when step 25℃is highlighted, click option, set Ramp as 0.1℃/sec)25 ℃, 5 min4 ℃,5 minPut on ice, or keep in -20℃ freezer for storage.3. Digest lentiCRISPR Dual Puro (or lentiCRISPR Dual GFP) with BsmBI and Topo-Dual with BbsIPurify the enzyme digested product with PCR product purification kit, and make sure that the concentration is more than 50 ng/ul.4. LigationDilute the annealed target oligos 200 times for further ligationLigate for more than 2 hours at 16 ℃. (Overnight is better.)5. Transformation and identification of positive clonesPick single clones and culture for several hours in 37℃incubator, and perform PCR to identify the positive clones.For lentiCRISPR Dual Puro and lentiCRISPR Dual GFP, use Oligo-Forward and CAG-R as primers; for Topo-Dual, use Oligo-Forward and Topo-F as primers.6. Digest Guide RNA Target2 expressing cassette in the Topo-Dual and lentiCRISPR Dual Puro-Target1(or lentiCRISPR Dual GFP-Target1)7. Gel extraction and purificationFor EZ-GuideXH-T arget2, cut and purify the smaller DNA band about 360 bps; for the PX458M-Target1, cut the uniquemain band about 9300 bps.8. LigationLigate overnight at 16 ℃.9. Transformation and identification of positive clonesUse Oligo-Forward (target1) and CAG-R as primers, and the PCR product is about 600 bp.10. Plasmid extraction and sequencing validation.Culture the positive clone in 10 ml Amp+ LB medium, and extract plasmid.Use CAG-R , Topo-F as sequencing primer.IV. Assess knockout efficiency in 293FT cells (or other cells)1. Transfect lentiCRISPR Dual GFP plasmid carrying Guide RNA expressing cassettes in 293FT cells for 36-48 hours. For mouse genes, MEF, 3T3 and some other easy-transfect cells can be used.2. Detect the GFP Fluorescence through fluorescent microscopy or examine the transfection efficiency through Flow cytometer.3. Extract genomic DNA from transfected cells. And perform genotyping with these gDNA samples. If the mutant PCR band is obvious and strong enough, the pair of Guide RNAs can be regarded as efficient.V. Lentivirus production----Lentiviral Production Protocol----The day before transfection:The day before transfection, seed around 5×106 HEK293FT cells per 100mm petri dishes.The day of transfection:1. Check to make sure cells are at least 70% confluent before transfection2. Combine the appropriate amount of plasmid DNAs in a 1.5ml conical tube:For per100mm petri dish:10 μg Lentivirus backbone plasmid harboring inteterest gene4 μg pdR8.2. (structural vecto r)2 μg pMD2.G (envelope vector)16 μg in total3. Add filtered water to a final volume of 878μ1.4. Add 122μ1 2M CaCl2 and mix.5. Drop 1000ul of 2xHBS to the abvove mixture slowly. Note: Shaking the tube continuously to avoid generating large particles6. Sit mixture at room temperature for 2min.7. Remove old media and replace with 9 ml new completeculture media.8. Add all of mixture to the cells gently and evenly. Swirl the petri dish to distribute evenly and put it back to incubator.9. 8 hours later, Change media to remove the transfection reagent and replace with 12 ml new complete culture media.10. Generally, 72 hours after transfection , harvest cell culture supernatant containing lentivirus and filter through a 0.45μm filter.11. Concentrate the lentivirus with PEG8000 or with ultra-speed centrifugation.Transfection reagent stock preparation:2 M CaCl22xHBS: 50mM HEPES10mM KCl12mM Dextrose280mM NaCl1.5mM Na2HPO4Resuspend in 900 ml H2O, adjust pH to 7.04 (with NaOH if using HEPES free acid, with HCl if using HEPES sodium salt). Raise volume to 1000 ml and carefully re-adjust final pH to 7.05 exactly (Note: pH needs to be very precise at pH 7.04 –7.05). Filter through 0.22μM and store in aliquots at -20°C (good for up to 6 months).Suggestions and Tips: You can also choose other transfection reagents including lipofectamine2000, PEI and Turbofect (Thermo/Fermentas). Remember that the transfection efficiency should be over 90 percent.VI. Knockout in interested cellsTheoretically, any cells transfected with plasmid should be KO cells. However, to get pure KO clones, single clones culture are necessary. Culture of single clones is a little difficult for some cell types. To improve the expansion of single cells, gelatin pre-coating and conditional medium can increase the survival rate of single cells.1. Plate the cells in 6-well plate one day before, and make sure the cell confluency reach about 70-80%.2. Transfect the interested cells with validated px459M based plasmid and culture for 24 hours.3. Treat the transfected cells with 2ug/ml puromycin for 3 or4 days. Set one mock un-transfected well as control.Cautions!: before treatment, a pilot experiment is needed to explore the optimized puromycin concentration.4. Withdraw the puromycin, and culture the cells with 20% FBS medium.5. Several days later, if independent clones expand, pick the clones and transfer them into 24-well plate for culture. The remained cells keep growing until about 50% cell confluency.6. When the cells in 24-well plate reach more than 30% confluency, trypsin-digest these wells, take out 80% cells for genotyping, and remain 20% cells for further culture.7. Quick-spin the cell suspension at 14000rpm for 30s. Remove the supernatant, and lyse the cell pellet with 20-60ul solution A. Heat the lysis at 95 ℃ for 10 min, then add the same volume solution B, vortex for a while, and centrifuge at 13000rpm for 10min. remove the supernatant for PCR template.8. Do the genotyping, and keep the clones containing knocked-out cells9. When the clones containing knocked-out cells reach full confluency, digest the cells with trypsin, and count the cell number. Serially dilute the cells in 40 cells/10ml medium by 10-fold.10. The medium for the step 9 should be the conditional medium. That is, coll ecting medium supernatant of the same cell type (culture time is less than or about 24 hours, avoiding the over-proliferating’s medium supernatant). Add 20% FBS in this supernatant and filter the medium with 0.22 ul filter.11. Take out 96-well plates, add 100 ul autoclaved 0.1% gelatin solution per well, then incubate in 37 ℃ for at least 30 min.12. Aspirate the gelatin, and aliquot 100 ul diluted 0.4 cell containing conditional medium per well. Each 8 or 10 wells are added, shake the cell suspension and continue.13. Add another 100ul conditional medium into the 96-well plate on the fourth or fifth day, and no later than the fifth day.14. When the cell confluency reach more than 30%, or the clones expand large enough, digest the cells and transfer them into 24-well plate, repeat the step6-8. Keep pure knocked-out clones for further experiment.15. For the remained cells in step 5. When cell reach 50% confluency, they can also be used for limiting dilution for single clones. Or this mixture can be frozen for later isolation.VII. Assess off targets effectOnce some clones are identified KO ones. The potential off-target effect should be detected. Website (/.) will list all potential off-targets site. Amplify those high-risky off-target site, andexamine whether off-target occur through sanger sequency. If any obvious off-targets are not detected, then these clones can be used for further functional experiment.P.S. Method for quick extraction cell genomic DNA1.Collect about 2×105 cells, and discard the supernatant.2.Add 75 μl 25mM NaOH/0.2mM EDTA.(Solution A).3.Place in thermocycler at 98℃ for 1 hours, then reduce the temperature to 15 ℃Until ready to proceed to the next step.4. Add 75 μl of 40mM Tris HCl(pH5.5).(Soluti on B)5. Centrifuge at 4000rpm for 3 minutes.6. Take an aliquot for PCR (use 2 μl of a 1:100 dilution/reaction).。
Phylogeny of Cas9determines functionalexchangeability of dual-RNA and Cas9among orthologous type II CRISPR-Cas systemsInes Fonfara 1,2,Anaı¨s Le Rhun 1,2,Krzysztof Chylinski 1,3,Kira S.Makarova 4,Anne-Laure Le´crivain 1,Janek Bzdrenga 1,Eugene V.Koonin 4and Emmanuelle Charpentier 1,2,5,*1The Laboratory for Molecular Infection Medicine Sweden (MIMS),Umea˚Centre for Microbial Research (UCMR),Department of Molecular Biology,Umea˚University,Umea ˚S-90187,Sweden,2Helmholtz Centre for Infection Research,Department of Regulation in Infection Biology,Braunschweig D-38124,Germany,3Deptartment of Biochemistry and Cell Biology,Max F.Perutz Laboratories,University of Vienna,Vienna A-1030,Austria,4National Center for Biotechnology Information,National Library of Medicine,NationalInstitutes of Health,Bethesda,MD 20894,USA and 5Hannover Medical School,Hannover D-30625,GermanyReceived September 11,2013;Revised September 30,2013;Accepted October 14,2013ABSTRACTThe CRISPR-Cas-derived RNA-guided Cas9endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms.The DNA-target-ing mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA),which directs Cas9to cleave invading DNA in a sequence-specific manner,dependent on the presence of a Protospacer Adjacent Motif (PAM)on the target.We show that evolution of dual-RNA and Cas9in bacteria produced remarkable sequence diversity.We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9and dual-RNA.We demonstrate that these two compo-nents are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated parison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9phylogeny corroborates horizontal transfer of the CRISPR-Cas loci.The reported collection of dual-RNA:Cas9with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9tool.INTRODUCTIONEditing genomes using the RNA-guided DNA targeting principle of CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated has widely the past few months (1–13).The main advantage provided by the bacterial type II CRISPR-Cas system lies in the minimal requirement for programmable DNA interference:an endonuclease,Cas9,guided by a custom-izable dual-RNA structure (14).As initially demonstrated in the original type II system of Streptococcus pyogenes ,trans -activating CRISPR RNA (tracrRNA)(15,16)binds to the invariable repeats of precursor CRISPR RNA (pre-crRNA)forming a dual-RNA (14–17)that is essential for both RNA comaturation by RNase III in the presence of Cas9(15–17),and invading DNA cleavage by Cas9(14,17–19).As demonstrated in Streptococcus ,Cas9guided by the duplex formed between mature activating tracrRNA and targeting crRNA (14–16)introduces site-specific double-stranded DNA (dsDNA)breaks in the invading cognate DNA (14,17–19).Cas9is a multi-domain enzyme (14,20,21)that uses an HNH nuclease domain to cleave the target strand (defined as complemen-tary to the spacer sequence of crRNA)and a RuvC-like domain to cleave the non-target strand (14,22,23),enabling the conversion of the dsDNA cleaving Cas9into a nickase by selective motif inactivation (2,8,14,24,25).DNA cleavage specificity is determined by two parameters:the variable,spacer-derived sequence of*To whom correspondence should be addressed.Tel:+4953161815500;Fax:+495316181805501;Email:emmanuelle.charpentier@helmholtz-hzi.deThe authors wish it to be known that,in their opinion,the first two authors should be regarded as Joint First Authors.Nucleic Acids Research,2013,1–14doi:10.1093/nar/gkt1074ßThe Author(s)2013.Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (/licenses/by/3.0/),which permits unrestricted reuse,distribution,and reproduction in any medium,provided the original work is properly cited.Nucleic Acids Research Advance Access published November 22, 2013 at Peking University on January 1, 2014/Downloaded fromcrRNA targeting the protospacer sequence (a protospacer is defined as the sequence on the DNA target that is complementary to the spacer of crRNA)and a short sequence,the Protospacer Adjacent Motif (PAM),located immediately downstream of the protospacer on the non-target DNA strand (14,18,23,26–28).Recent studies have demonstrated that RNA-guided Cas9can be employed as an efficient genome editing tool in human cells (1,2,8,11),mice (9,10),zebrafish (6),drosophila (5),worms (4),plants (12,13),yeast (3)and bacteria (7).The system is versatile,enabling multiplex genome engineering by programming Cas9to edit several sites in a genome simultaneously by simply using multiple guide RNAs (2,7,8,10).The easy conversion of Cas9into a nickase was shown to facilitate homology-directed repair in mammalian genomes with reduced mutagenic activity (2,8,24,25).In addition,the DNA-binding activity of a Cas9catalytic inactive mutant has been exploited to engineer RNA-programmable transcrip-tional silencing and activating devices (29,30).At present,RNA-guided Cas9from S.pyogenes ,S.thermophilus**and Neisseria meningitidis have been de-veloped into tools for genome manipulation (1–13,24,25,31–34).Here,we explore the possibilities of ex-panding the RNA-programmable Cas9toolbox to add-itional orthologous systems.We investigated the diversity and interchangeability of dual-RNA:Cas9in eight representatives of phylogenetically defined type II CRISPR-Cas groups.The results of this work not only introduce a wider range of Cas9enzymes,dual-RNA structures and associated specific PAMs but also enlighten the evolutionary aspects of type II CRISPR-Cas systems,including coevolution and horizontal transfer of the system components.MATERIALS AND METHODS Bacterial strains and culture conditionsSupplementary Table S1lists bacterial strains used in this study.Streptococcus pyogenes ,Streptococcus mutans ,Campylobacter jejuni ,N.meningitidis ,Escherichia coli and Francisella novicida were grown as previously described (15,16).Brain Heart Infusion (BHI,Becton Dickinson)agar and BHI broth medium supplemented with 1%glucose and 1%lactose were used to culture S.thermophilus at 42 C in a 5%CO 2environment (16).Pasteurella multocida and Staphylococcus aureus were grown at 37 C on BHI agar plates and in BHI broth with shaking.Cell growth was monitored by measuring the optical density of cultures at 620nm (OD 620)using a microplate reader (BioTek PowerWave).Bacterial transformationEscherichia coli was transformed with plasmid DNA according to standard protocols (35).Transformation of S.pyogenes was performed as previously described (36)with some modifications.Streptococcus pyogenes pre-cultures were diluted 1:100in fresh THY medium and grown at 37 C,5%CO 2until OD 620reached 0.3.Glycine was added to the medium to 10%finalconcentration and growth was maintained for an add-itional hour.Cells were spun down at 4 C at 2500Âg and washed three times with electroporation buffer (5mM KH 2PO 4,0.4M D-sorbitol,10%glycerol,pH 4.5),finally suspended in the same buffer and equalized to the same OD 620.For electroporation,1m g of plasmid was incubated with the competent cells on ice for 10min.The conditions were 25m F,600V and 1.5V using 1mm electroporation cuvettes (Biorad).After a regeneration time of 3h,bacteria were spread on agar medium supple-mented with kanamycin (300m g/ml).Transformation assays were performed at least three times independently with technical triplicates.The efficiencies were calculated as colony-forming units (CFU)per m g of plasmid DNA.Positive and negative control transformations were done with backbone plasmid pEC85and sterile H 2O,respectively.DNA manipulationsDNA manipulations including DNA preparation (QIAprep Spin MiniPrep Kit,Qiagen),polymerase chain reaction (PCR)(Phusion ÕHigh-Fidelity DNA Polymerase ,Finnzyme),DNA digestion (restriction enzymes,Fermentas),DNA ligation (T4DNA ligase,Fermentas),DNA purification (QIAquick PCR Purification Kit,Qiagen)and agarose gel electrophoresis were performed according to the standard techniques or manufacturers’protocols with some modifications (35).Site-directed mutagenesis was done using QuikChange II XL kit (Stratagene)or PCR-based mutagenesis (37).Synthetic oligonucleotides (Sigma-Aldrich and Biomers)and plasmids used and generated in this study are listed in Supplementary Table S1.The integrity of all con-structed plasmids was verified by enzymatic digestion and sequencing at LGC Genomics.Construction of plasmids for complementation studies in S.pyogenesThe backbone shuttle vector pEC85was used for comple-mentation study (38,39).The RNase-III encoding genes (rnc genes)of S.pyogenes ,S.mutans ,S.thermophilus ,C.jejuni ,N.meningitidis ,P.multocida , F.novicida ,E.coli and S.aureus ,and the genes encoding truncated and inactive RNase III variants (truncated and inactive (D51A)rnc mutants)of S.pyogenes were cloned in pEC483(pEC85containing the native promoter of S.pyogenes rnc )using NcoI and EcoRI restriction sites (Supplementary Table S1,Supplementary Figure S6).The orthologous and mutant cas9genes were cloned in pEC342(pEC85containing a sequence encoding tracrRNA-171nt (16)and the native promoter of the S.pyogenes cas operon)using SalI and SmaI restriction sites (Supplementary Table S1).Note that in a previous study,we observed low abundance of tracrRNA in the cas9deletion mutant.For this reason,plasmids used in cas9complementation studies were designed to encode tracrRNA in addition to cas9(16).The generated rnc and cas9recombinant plasmids were introduced in S.pyogenes Árnc and Ácas9deletion strains,respectively (Supplementary Table S1).Plasmid integrity in all2Nucleic Acids Research,2013at Peking University on January 1, 2014/Downloaded fromcomplemented strains was checked by plasmid DNA extraction and digestion.Construction of plasmids for transformation studies inS.pyogenesPlasmid pEC85was used as backbone vector for trans-formation studies.A DNA fragment containing WT speM protospacer sequence was cloned in the PstI site of plasmids containing coding sequences of WT or mutated cas9from S.pyogenes(Supplementary Table S1). Construction of plasmids for protein purificationThe overexpression vector pET16b(Novagen)was modified by inserting three additional restriction sites (SalI,SacI,NotI)into the NdeI restriction site,generating pEC621.The genes coding for the orthologous Cas9 proteins were PCR amplified from genomic DNA of the corresponding strains using primers containing a SalI and a NotI restriction site(Supplementary Table S1).The S.pyogenes cas9mutant genes were PCR amplified from the complementation plasmids mentioned above.All orthologous and mutant cas9genes were cloned into the SalI and NotI sites of pEC621.Construction of substrate plasmids for in vitrocleavage assaysPlasmid pEC287that contains the speM protospacer sequence was used as a vector to construct all substrate plasmids.The PAM sequence located in30just next to the crRNA-targeted sequence of the speM protospacer(GGG on this plasmid)was modified by PCR-mediated site-directed mutagenesis(37)using one standard oligonucleo-tide(OLEC3140or OLEC3194)that either introduced or removed a XbaI restriction site for screening purposes, and a second mutagenic oligonucleotide to exchange the protospacer adjacent sequence(Supplementary Table S1). RNA preparationTotal RNA from S.pyogenes SF370WT,deletion mutants and complemented strains was prepared from culture samples collected at the mid-logarithmic phase of growth using TRIzol(Sigma-Aldrich).The total RNA samples were treated with DNase I(Fermentas)according to the manufacturer’s instructions.The concentration of RNA in each sample was measured using NanoDrop. Northern blot analysisNorthern blot analysis was carried out essentially as described previously(40–42).Total RNA was separated on10%polyacrylamide8M urea gels and further processed for blotting on nylon membranes(Hybond TM N+,GE healthcare;Trans-BlotÕSD semi-dry transfer apparatus,Biorad;1X TBE,2h at10V/cm),chemical cross-linking with EDC(1-Ethyl-3-(3-dimethyla-minopropyl)carbodiimide hydrochloride)(41)and prehybridization(Rapid-hyb buffer,GE healthcare;1h at42 C).Oligonucleotide probes(40pmol)were labeled with32P(20m Ci)using the T4-polynucleotide kinase (10U,Fermentas)and purified using G-25columns (GE Healthcare)prior use.Visualization of the radio-active signal was done using a phosphorimager.5SrRNA served as loading control.Protein purificationEscherichia coli Rosetta2(DE3)and E.coli NiCo21(DE3) (New England Biolabs)were transformed with overexpression plasmids coding for S.pyogenes WT and mutant or orthologous Cas9,respectively.Cells were grown at37 C to reach an OD600of0.7–0.8,protein expression was induced by adding IPTG to afinal concen-tration of0.5mM and cultures were further grown at13 C overnight.The cells were harvested by centrifugation andthe pellet was resuspended in lysis buffer(20mM HEPES,pH7.5,500mM KCl[1M for S.thermophilus*Cas9],0.1%Triton X-100,25mM imidazole)and lysed by son-ication.The lysate was cleared by centrifugation(>20000Âg)and incubated with Ni-NTA(Qiagen)for1h at4 C.After washing the Ni-NTA with lysis bufferand wash buffer(20mM HEPES,pH7.5,300mM KCl,0.1%Triton X-100,25mM imidazole),the recombinant protein was eluted with elution buffer(20mM HEPES,pH7.5,150mM KCl,0.1mM DTT,250mM imidazole,1mM(EDTA))and the fractions were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE).In the case of S.pyogenesCas9WT and mutants,the protein containing eluateswere pooled and further purified via HiTrap SP FF(GE Healthcare)cation-exchange chromatography.Briefly,the protein was loaded on the column equilibrated with bufferA(20mM HEPES pH7.5,100mM KCl)using an FPLC system(A kta,GE Healthcare).Cas9was eluted with a gradient of buffer B(20mM HEPES pH7.5,1M KCl)over12ml.1ml fractions were collected and analyzed by (SDS–PAGE).The protein containing fractions were pooled and dialyzed overnight(20mM HEPES,pH7.5,150mM KCl,50%glycerol).For Cas9orthologs,the eluates from Ni-NTA purification were checked forpurity by SDS–PAGE.In case of contaminants,a second purification over chitin beads was performed as described in the manual for NiCo21(DE3)cells fromNew England Biolabs.Briefly,1ml chitin beads(New England Biolabs)equilibrated with buffer A was incubated with the Ni2+-IMAC eluates for1h at4 C. Afterwards,the beads were added onto a column andthe Cas9containingflowthroughs were collected andagain checked for purity by SDS–PAGE(Supplementary Figure S1).The purified proteins were dialyzed overnight.The protein concentration was calculated by measuringthe OD280using the extinction coefficient.The detailed characteristics of purified proteins are summarized in Supplementary Figure S1A.In vitro transcriptionRNA for in vitro DNA cleavage assays was generatedby in vitro transcription using the AmpliScribe TM T7-Flash TM Transcription Kit(Epicentre)according to the manufacturer’s instructions.PCR products or synthetic oligonucleotides used as templates are listed in Supplementary Table S1.The synthesized tracrRNA andNucleic Acids Research,20133at Peking University on January 1, 2014/Downloaded fromrepeat region of crRNA from each bacterial species cor-respond to the mature forms of RNAs as determined by deep RNA sequencing (15)or bioinformatics predictions.The spacer region of all crRNAs used in this study targets the speM protospacer (encoding superantigen;targeted by spacer 2of S.pyogenes SF370CRISPR array,Spyo1h_002(16)).Transcribed RNAs were precipitated and further purified from 10%polyacrylamide 8M urea denaturing gel.The RNA concentration was determined by measuring the OD 260and the molarity was calculated.Equimolar amounts of crRNA and tracrRNA were mixed in 5X RNA annealing buffer (1M NaCl,100mM HEPES,pH 7.5),heated up to 95 C for 5min and slowly cooled to room temperature before use.In vitro DNA cleavage assaysFor the cleavage assays using Cas9mutant proteins,25nM of Cas9were incubated with equimolar amounts of prehybridized S.pyogenes dual-RNA in cleavage buffer (20mM HEPES,pH 7.5,150mM KCl,10mM MgCl 2,0.5mM DTT,0.1mM EDTA)for 15min at 37 C.Plasmid DNA (5nM)containing speM (NGG PAM)was added and further incubated for 1h at 37 C.The reaction was stopped by addition of 5X loading buffer (250mM EDTA,30%glycerol,1.2%SDS,0.1%(w/v)bromophenol blue)and analyzed by 1%agarose gel elec-trophoresis in 1X TAE.Cleavage products were visualized by ethidium bromide staining.All other cleavage assays were carried out using the same conditions with the fol-lowing modifications:KGB (43)(100mM potassium glu-tamate,25mM Tris/acetate,pH 7.5,10mM Mg-acetate,0.5mM 2-mercaptoethanol,10m g/ml bovine serum albumin)was used as cleavage buffer and different con-centrations of dual-RNA:Cas9complex were analyzed.The concentration of plasmid DNA was kept constant in all experiments,i.e.5nM.Search for PAM motifsSpacer sequences of the selected bacterial species were ex-tracted from the CRISPRdatabase (http://crispr.u-psud.fr/crispr/)and used to find cognate protospacer candidates using megaBLAST (/Blast).Protospacer candidates were defined as containing a sequence with !90%similarity to the crRNA spacer sequence and originating from phage,plasmid or genomic DNA related to the bacterial species of the tar-geting CRISPR-Cas.For the investigated CRISPR-Cas loci,the orientation of transcription was determined pre-viously by RNA sequencing or northern blot analysis (15,16).It was also shown before that in type II CRISPR-Cas,the PAM sequence is located in 30of the protospacer,juxtaposed to the sequence targeted by cognate crRNA on the non-target strand (14,18,23,44).To identify possible PAMs in each bacterial species,10nt sequences on the non-target strand directly down-stream of each protospacer sequence were aligned.A logo plot (/)showing the most abundant nucleotides was created and PAM sequences were predicted.In the cases of CRISPR-Cas loci for which no suitable protospacer sequences could beidentified (S.mutans UA159, C.jejuni NCTC 11168,P.multocida Pm70, F.novicida U112),closely related strains of the same species were selected (Supplementary Table S2).The spacer contents of the type II CRISPR arrays in selected strains were analyzed (http://crispr.u-psud.fr/Server/).The spacer sequences were then used to select cognate protospacer sequences as described above.Protein sequence analysisPosition-Specific Iterated (PSI)-BLAST program (45)was used to retrieve orthologs of the Cas9family in the NCBI nr database.Sequences shorter than 800amino acids were discarded.The BLASTClust program (46)set up with a length coverage cutoff of 0.8and a score coverage thresh-old (bit score divided by alignment length)of 0.8was used to cluster the remaining sequences (Supplementary Table S2).This procedure produced 82clusters.In the case of sequences reported in this study,one or several represen-tatives from each cluster were selected and aligned using the MUSCLE program (47)with default parameters,followed by a manual correction on the basis of local alignments obtained using PSI-BLAST (45)and HHpred programs (48).The confidently aligned blocks (Supplementary Figure S2)with 285informative positions were used for maximum likelihood tree reconstruction using the FastTree program (49)with the default param-eters:JTT evolutionary model,discrete gamma model with 20rate categories.The same program was used to calculate the bootstrap values.Cas1sequences were selected from the corresponding cas operons (Supplementary Table S2).A few incomplete sequences were substituted by other Cas1sequences from the same Cas9cluster (Supplementary Table S2).Several Cas1proteins from subtypes I-A,B,C and E were included as an outgroup.Cas1sequences were aligned using the same approach described above and 252informative positions (Supplementary Figure S3)were used for maximum like-lihood tree reconstruction using the FastTree program.RNase III multiple sequence alignment was prepared using the MUSCLE program.RNA sequence and structure analysisRNA duplex secondary structures were predicted using RNAcofold of the Vienna RNA package (50,51)and RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/).The structure predictions were then visualized using VARNA (52).RESULTSDiversity of Cas9orthologsTo investigate the evolution and diversity of dual-RNA:Cas9systems,we subjected publicly available genomes to multiple rounds of BLAST search using pre-viously retrieved Cas9sequences as queries (15).Cas9orthologs were identified in 653bacterial strains represent-ing 347species (Supplementary Table S2).After removing incomplete or highly similar sequences,we selected 83diverse,representative Cas9orthologs for multiple4Nucleic Acids Research,2013at Peking University on January 1, 2014/Downloaded fromsequence alignment and phylogenetic tree reconstruction (Figure1A,Supplementary Table S2,Supplementary Figures S2and S4,see‘Materials and Methods’section). The Cas9tree topology largely agrees with the phylogeny of the corresponding Cas1proteins(Supplementary Table S2,Supplementary Figures S3and S4)and fully supports the previously described classification of type II CRISPR-Cas into three subtypes,II-A(specified by csn2),II-B (characterized by long and most diverged cas9variants (formerly csx12)and cas4)and II-C(three-cas gene operon)(15).Analysis of the composition of cas genes,transcription direction of the CRISPR arrays with respect to that of the cas operon,and location and orientation of tracrRNAs resulted in the division of subtypes into groups with distinct locus characteristics,especially within the subtype II-A(Figure1,clusters marked with different colors)(15).We selected Cas9enzymes representative of the major type II groups.Cas9orthologs of S.pyogenes, S.thermophilus*(CRISPR3)and S.mutans were chosen for type II-A systems associated with shorter,$220amino acid Csn2variants(Csn2a).Cas9of S.thermophilus** (CRISPR1)represents a distinct group of type II-A sequences associated with longer,$350amino acid version of Csn2orthologs(Csn2b).Cas9of F.novicida was selected for type II-B.The closely related Cas9 orthologs of P.multocida and N.meningitidis and the distinct,short Cas9of C.jejuni were chosen for type II-C(Figure1B).Expression of associated tracrRNAs and crRNAs in S.pyogenes,S.mutans, F.novicida, N.meningitidis and C.jejuni was already validated by deep RNA sequencing(15,16).The RNAs in S. thermophilus and P.multocida were predicted bioinformatically based on the sequences from related species within the same type II group.Figure1B shows the organization of the eight selected type II CRISPR-Cas loci and highlights our previousfindings demonstrating that the type II loci architectures are highly variable among subtypes,yet conserved within each group(15). These variations are in good agreement with the clustering derived from the Cas9and Cas1phylogenetic trees (Figure1A,Supplementary Figure S4).Bacterial RNases III are interchangeable in dual-RNA maturationAs described in S.pyogenes and S.thermophilus,RNase III plays an essential role in the biogenesis of dual-RNA:Cas9systems by coprocessing tracrRNA and pre-crRNA at the level of antirepeat:repeat duplexes(16,17). We analyzed the interchangeability of S.pyogenes RNase III with RNases III from selected bacterial species in the coprocessing of S.pyogenes tracrRNA:pre-crRNA, including strains that lack type II CRISPR-Cas (S.aureus COL,E.coli TOP10).Northern blot analysis shows that all RNases III studied here can coprocess the RNA duplex(Figure2,Supplementary Figure S5), indicating that there is no species-specificity for tracrRNA:pre-crRNA cleavage by RNase III.Multiple sequence alignment of RNase III orthologs demonstrates conservation of the catalytic aspartate residue andthe Figure1.Phylogeny of representative Cas9orthologs and schematic representation of selected bacterial type II CRISPR-Cas systems.(A)Phylogenetic tree of Cas9reconstructed from selected,informative positions of representative Cas9orthologs multiple sequence alignmentis shown(Supplementary Figure S2and Supplementary Table S2).TheCas9orthologs of the subtypes classified as II-A,II-B and II-C are highlighted with shaded boxes.The colored branches group distinctNucleic Acids Research,20135(continued)at Peking University on January 1, 2014/Downloaded fromdsRNA binding domain (Figure 2,Supplementary Figure S6)that are both required for RNA coprocessing (Figure 2,Supplementary Figure S5).These data imply that the conservation of tracrRNA:pre-crRNA coprocess-ing by bacterial RNase III provides a degree of flexibility allowing the functionality of dual-RNA:Cas9systems in multiple species upon horizontal transfer.Cas9HNH and split RuvC domains are the catalytic moieties for DNA interferenceComparison of Cas9sequences revealed high diversity in amino acid composition and length (984amino acid for C.jejuni to 1648amino acids for F.novicida ),especially in the linker sequence between the highly conserved N-terminal RuvC and central RuvC-HNH-RuvC regions and in the C-terminal extension (Supplementary Figure S2).Several studies demonstrated the importance of the nuclease motifs for dsDNA cleavage activity by mutating one aspartate in the N-terminal motif of the RuvC domain and one or several residues in the predicted catalytic motif of the HNH domain of the Cas9enzyme (14,22,23).To investigate the relevance of all catalytic motifs for tracrRNA:pre-crRNA processing and/or DNA interfer-ence,alanine substitutions of selected residues were created (Figure 3A).In addition to the already published catalytic amino acids,we created Cas9point mutants of conserved amino acid residues in the central RuvC motifs (14)(Figure 3A,Supplementary Figure S2).Northern blot analysis of S.pyogenes cas9deletion mutant comple-mented with each of the cas9point mutants revealed thepresence of mature tracrRNA and crRNA forms,demonstrating that none of the catalytic motifs is involved in dual-RNA maturation by RNase III.This is in agreement with previous data showing that RNase III is the enzyme that specifically cleaves tracrRNA:pre-crRNA duplex (16).Cas9seems to have a stabilizing function on dual-RNA.We show that the catalytic motifs are not involved in RNA duplex stabilization (Figure 3B,Supplementary Figure S7).To investigate the involvement of the conserved motifs of Cas9in DNA interference in vivo ,we used a previously described plasmid-based read-out system that mimics infection with invading protospacer-containing DNA elements (16).Transformation assays were done in S.pyogenes WT or a cas9deletion mutant using plasmids containing the speM protospacer gene (comple-mentary to the second spacer of S.pyogenes SF370type II CRISPR array (16))and WT or mutant cas9(Figure 3C).In this assay,Cas9expressed following plasmid delivery in bacterial cells catalyzes its own vector cleavage,when active.Control experiments showed that the speM protospacer-containing plasmid was not tolerated in WT S.pyogenes ,demonstrating activity of WT CRISPR-Cas.Similarly,a plasmid containing the speM protospacer and encoding WT Cas9could not be maintained in the cas9deletion mutant,demonstrating that Cas9is able to cleave the plasmid from which it is expressed.Except for Cas9N854A,all plasmids encoding Cas9mutants were tolerated in the cas9deletion strain,indicating abrogation of Cas9interference activity for these variants.The in vivo DNA targeting data were confirmed with in vitro DNA cleavage assays.Purified WT and mutant Cas9proteins were incubated with tracrRNA:crRNA tar-geting speM and subjected to cleavage of plasmid DNA containing the speM protospacer.WT and N854A Cas9show dsDNA cleavage activity,whereas the other Cas9mutants cleave only one strand of the dsDNA substrate,yielding nicked open circular plasmid DNA (Figure 3D).This corroborates the results obtained in vivo showing the importance of the conserved nuclease motifs for DNA interference by Cas9.In addition to the previously pub-lished data demonstrating the importance of the N-terminal RuvC motif and the catalytic motif of HNH,we thus defined new catalytic residues in the central RuvC motifs.Only Cas9from closely related CRISPR-Cas systems can substitute for S.pyogenes Cas9in tracrRNA-directed pre-crRNA maturation by RNase IIIBeside the conservation of the HNH and split RuvC domains involved in DNA cleavage (14,15),the length of Cas9orthologs and the amino acid sequences of Cas9are highly variable among the different groups of type II CRISPR-Cas systems (Figure 4A,Supplementary Figure S2).Hence,we investigated whether this variability plays a role in the specificity of Cas9with regard to tracrRNA:pre-crRNA duplex and mature crRNA stabil-ization.We complemented S.pyogenes cas9deletion mutant with Cas9from selected bacterial species represen-tative of the various type II groups and analyzedFigure 1.Continuedproteins of closely related loci with similar locus architecture (15).Each protein is represented by the GenInfo (GI)identifier followed by the bacterial strain name.The bootstrap values are given for each node (see ‘Materials and Methods’section).Note that the monophyletic clusters of subtypes II-A and II-B are supported by high bootstrap values.The scale bar for the branch length is given as the estimated number of amino acid substitution per site.(B )Genetic loci of type II (Nmeni/CASS4)CRISPR-Cas in Streptococcus pyogenes SF370,Streptococcus mutans UA159,Streptococcus thermophilus LMD-9*(CRISPR3),**(CRISPR1),Campylobacter jejuni NCTC 11168,Neisseria meningitidis Z2491,Pasteurella multocida Pm70and Francisella novicida U112.Red arrow,transcription direction of tracrRNA;blue arrows,cas genes;black rectangles,CRISPR repeats;green diamonds,spacers;thick black line,leader sequence;black arrow,putative pre-crRNA promoter;HP,Hypothetical Protein.The colored bars represented on the left correspond to Cas9tree branches colors.The transcription direction and putative leader position of C.jejuni and N.meningitidis pre-crRNAs were derived from previously published RNA sequencing data (15).The CRISPR-Cas locus architecture of P.multocida was predicted based on its close similarity to that of N.meningitidis and further confirmed by bioinformatics prediction of tracrRNA based on a strongly predicted promoter and a transcrip-tional terminator as described in (15).Type II CRISPR-Cas loci can differ in the cas gene composition,mostly with cas9,cas1and cas2being the minimal set of genes (type II-C,blue),sometimes accompanied with a fourth gene csn2a /b (type II-A,yellow and orange)or cas4(type II-B,green).The CRISPR array can be transcribed in the same (type II-A,yellow and orange)or in the opposite (types II-B and C,blue and green)direction of the cas operon.The location of tracrRNA and the direction of its transcription differ within the groups (compare type II-A of S.thermophilus **with type II-A from the other species indicated here (yellow)and compare type II-C of C.jejuni with type II-C of N.meningitidis and P.multocida (blue)).6Nucleic Acids Research,2013at Peking University on January 1, 2014/Downloaded from。
