合成子的形成的制作方法

背景技术：
：合成生物学依赖于由组分部分构建新的dna的能力。双链(ds)dna分子已通过在第一dna双链的两端产生交错末端得以组装。使用限制性核酸内切酶、或者通过使用核酸外切酶消化、或者通过t4dna聚合酶，然后杂交，以及任选地将第二dna双链与第一双链连接，已经实现这一点。在反应混合物中使用核酸外切酶和连接酶的技术还指定使用非链置换聚合酶。技术实现要素：除了其它，本公开提供一种组合物，其包括：i.5’-3’核酸外切酶；ii.链置换聚合酶；iii.单链(ss)dna结合蛋白质；和iv.非天然存在的缓冲剂，其中组合物不包括拥挤剂和/或非链置换聚合酶。该组合物可以用于将多核苷酸组装到合成子(synthon)中。取决于出于在包含其自身连接酶的细菌细胞中克隆的目的进行组装，还是出于不包括细菌中克隆步骤的目的进行组装，组合物的实施方式任选地包含连接酶。在先描述的组装方法需要非链置换聚合酶，并进一步额外地需要拥挤剂(crowdingagent)(例如，参见us8,968,999)。与现有技术相反，本发明的实施方式表明，当与5’-3’核酸外切酶一起使用时，链置换聚合酶相对于非链置换聚合酶具有优势。相对于包括拥挤剂，这一组合倾向于包括ss结合蛋白质，这与声称拥挤剂的使用比包括使用单链结合蛋白质的替代方案要有效4倍的现有技术教导相反。本发明的实施方式提供组合物、方法和试剂盒，其在单步方法中和/或在单一反应容器中提供由包括ds和/或ss核酸分子的寡核苷酸和多核苷酸来组装和克隆功能性基因和合成子的效率得以增加。这些实施方式不依赖于拥挤剂，也不需要非链置换聚合酶用于填充两分子退火之后存在的间隙。例如，当作用于dsdna的5’-3’核酸外切酶产生可以与来自另一分子的3’ssdna突出端有效退火的3’ssdna突出端时，链置换聚合酶可以填充分子退火之后留下的间隙。链置换dna聚合酶和5’-3’核酸外切酶活性的组合产生在接合位点处或附近包含切口的双链合成子。这一切口可以通过连接酶在体外密封，或者通过内源性细胞连接酶在体内密封。此外，在反应混合物中包括ssdna结合蛋白质使得能够有效地组装相对低浓度的核酸片段，从而在不损失效率或者不损失接合准确性的情况下节约成本。在一些组合物实施方式中，链置换聚合酶是b族聚合酶。链置换聚合酶应当优选地在相同反应条件下(例如，使用比如图1a～1e中所述的试验)链置换活性大于以聚合酶(thermofisher，waltham，ma)(通常描述为非链置换的)所观察者。在本发明的组合物、方法和试剂盒中，主要使用链置换聚合酶的链置换活性。在一些实施方式中，链置换聚合酶可以是非天然存在的，例如，链置换聚合酶可以是突变体。突变体的实例包括具有一个或更多个氨基酸替换的聚合酶，非天然存在的聚合酶可以或者另外是具有不相关氨基酸序列的部分的融合蛋白，其中融合聚合酶在自然界不存在。优选地，链置换聚合酶在50℃或更高下稳定，并且因此可以称作热稳定性链置换聚合酶。在一些情形中，链置换聚合酶是具有不相关或异源性dna结合结构域的融合聚合酶。在一些实施方式中，聚合酶部分的氨基酸序列与seqidno:102的同一性可以为至少90％或95％或98％或99％。在另一个实施方式中，聚合酶的氨基酸序列与seqidno:1的同一性可以为至少90％或95％或98％或99％或100％，优选至少90％。在另一个实施方式中，聚合酶的氨基酸序列与seqidno:33至seqidno:55中任意者的同一性可以为至少90％或95％或98％或99％或100％，优选至少90％。在一些实施方式中，dna结合结构域部分的氨基酸序列与seqidno:2的同一性可以为至少90％或95％或98％或99％。在另一个实施方式中，聚合酶的氨基酸序列与seqidno:1、seqidno:3、seqidno:56至seqidno:96或seqidno:102中任意者的同一性可以为至少90％或95％或98％或99％或100％，优选至少90％。在另一个实施方式中，本文所述的任意聚合酶结构域部分可以与本文所述的任意dna结合结构域组合，其条件是聚合酶部分和dna结合结构域是异源性的。例如，在其他实施方式中，融合蛋白的氨基酸序列与seqidno:1和seqidno:2的同一性可以为至少90％或95％或99％或100％，优选至少90％。在其他实施方式中，融合蛋白与seqidno:3的序列同一性可以为至少90％或95％或98％或99％或100％，优选至少90％。链置换聚合酶可以具有或不具有3’-5’核酸外切酶活性。链置换聚合酶具有3’-5’核酸外切酶活性时，多核苷酸接合可以通过使用包括本文例示者的条件来平衡3’-5’核酸外切酶活性、5’-3’聚合活性和链置换活性加以优化。组装的效率和准确性可以使用本文所述的试验来确认(例如，参见图3a和3b)。在一些实施方式中，聚合酶不是phusion、9°n、pfu或vent或者氨基酸序列与phusion或野生型9°n、pfu或vent的同一性为至少90％的聚合酶。在一些实施方式中，聚合酶是热稳定性的，即，在至少40℃或至少50℃的温度下有活性。与链置换聚合酶相反，一些聚合酶比如taqdna聚合酶经由5’→3’核酸外切酶活性降解遇到的下游链。该活性用于切口翻译方案。因此taqdna聚合酶不包括在链置换聚合酶的定义中。确定合成子形成的效率和准确性的试验描述于实施例中，并示于图3a～b。设计的组装片段编码lacl和lacz蛋白质，如果dna片段组装正确，其产生蓝色菌落。因此，过夜板“蓝色”菌落的数目指示组装的效率和准确性。在不存在蓝色的情况下，可能发生有效的组装，但接合/延伸区的错误阻止了表达。当合成子得以组装、并且然后克隆到宿主细胞中，合成子形成的效率和准确性转化成每个克隆将包含正确组装的合成子的置信度。借助该置信度，仅需要对一个或多个复制的克隆进行测序，以确认合成子的存在。这样降低了对可能包含错误的克隆进行测序的成本和不便。在一个实施方式中，至少80％或者另外可选地至少90％的克隆将会包含正确组装的合成子。在一些实施方式中，使用本文所述组合物的方法能够达到基本上超过最小需求的产率。例如，在单一转化事件中可以制备至多5,000或10,000个克隆。如果组装的目的不是制备合成子文库，而是制备单例合成子，则可以使用更低的核酸片段和试剂起始量，甚至其低于本文提供的范围。适合用于组装混合物的浓度范围的实例包括以下：0.02nm～100nm的dna片段或者例如0.2nm～10nmdna可以加入到反应容器中的试剂混合物中。在一个实施方式中，尽管可以使用更高或更低的比例，载体dna与dna片段以1:1的比例包括在内。与对于dsdna所选择的浓度相比较，可以优选更高的ssdna浓度。反应容器中的试剂混合物还可以包括0.0004u/μl～0.064u/μl的5’-3’核酸外切酶(例如0.0004u/μl～0.01u/μl)；0.5u/μl～32u/μl任选的连接酶(例如1u/μl～10u/μl)；0.0025u/μl～0.25u/μl链置换聚合酶(例如0.005u/μl～0.1u/μl)；和0.001μg/μl～0.1μg/μl的ss结合蛋白质(例如0.01μg/μl～0.5μg/μl)(单位对应于制造商(newenglandbiolabs,ipswich,ma)所指定者)。5’-3’核酸外切酶的量能够根据核酸片段重叠长度和每个片段的大小加以进一步优化。例如，5’-3’核酸外切酶的量可以在核酸片段长度大于80个核苷酸的范围内增加。指定范围内链置换聚合酶的绝对浓度并非关键。尽管已知有许多其他的ssdna结合蛋白质，并且其可以用于组合物中，组合物中使用的ssdna结合蛋白质可以是大肠杆菌(e.coli)reca、t7基因2.5产物、redb(来自噬菌体λ)或rect(来自rac原噬菌体)、etssb(极端热稳定的单链dna结合蛋白质)或者与seqidno:100的序列同一性为90％的ss结合蛋白质。如通过菌落数目所测量，比起在否则不存在ss结合蛋白质的情况下所发生的，包括ss结合蛋白质提高了特别是对于具有更长重叠序列(例如，至少20个核苷酸)的核酸片段的组装效率。任选的连接酶可以是依赖于nad+的连接酶，比如taq连接酶，或者依赖于atp的连接酶，比如t4连接酶。然而，对于pcr，由于atp能够抑制后续的合成子扩增中使用的taq聚合酶，使用依赖于nad+的连接酶是方便的。合适的连接酶的实例包括与seqidno:101的序列同一性为至少90％的蛋白质。在此使用的5’-3’核酸外切酶可以是具有5’-3’核酸外切酶活性以及ss核酸内切酶活性的酶(参见，例如，garforth等人,pnas,96,38-43(1999))。具有核酸外切酶和ss核酸内切酶活性的5’-3’核酸外切酶的实例包括t5核酸外切酶、以及其同源物和变体。在一个实施例中，5’-3’核酸外切酶与seqidno:98的氨基酸序列同一性为至少90％。没有要求在用链置换聚合酶接合多核苷酸之前使5’-3’核酸外切酶变性。因此，在实施例中描述了使用热稳定性5’-3’核酸外切酶。在一些实施方式中，组合物还可以包括dntp(即，dgtp、datp、dgtp和dttp的混合物)，并且，在一些实施方式中，当使用t55’-3’核酸外切酶时，组合物还可以包括钾盐，比如kcl(例如，浓度范围7mm～150mm)。概括地讲，提供一种制备合成子的方法。在一些实施方式中，该方法可以包括在合适的反应条件下将本文所述组合物的一种实施方式与一组多核苷酸和/或寡核苷酸一起孵育，上述组合物的实施方式包括本文所述的链置换聚合酶和5’-3’核酸外切酶和任选的连接酶(如果反应在体外或者在不含连接酶的细胞或有机体中的体内)，并且还可以包含ss结合蛋白质，在上述的一组多核苷酸和/或寡核苷酸中至少一种或一些组成员具有与一种或一些其他组成员重叠的序列。在一些实施方式中，多核苷酸或寡核苷酸可以是dsdna，例如，重叠pcr产物或重叠限制片段。在其他实施方式中，多核苷酸可以是ssdna或rna。在一些实施方式中，该多核苷酸组可以包括ssdna或rna。在一些实施方式中，该多核苷酸组可以包括ds多核苷酸。在一些实施方式中，该多核苷酸组可以包括至少一种ds多核苷酸和至少一种ss多核苷酸。在一些实施方式中，该多核苷酸组可以包括除了在亚群成员之间变化的子序列以外具有相同序列的多核苷酸亚群。在其他实施方式中，该多核苷酸组可以包括ss或ds多核苷酸，或者在其用于接合目的的末端具有重叠区、但形成合成子的内部序列不同的多核苷酸。因此，在本发明的方法的一个实施方式中，该多核苷酸组中的多核苷酸为ds；比如，其中ds多核苷酸是重叠pcr产物或重叠限制片段或者由ss多核苷酸组装。在本发明的方法的一另外可选的实施方式中，合成子由该多核苷酸组中ss的多核苷酸组装。在本发明的方法的另一另外可选的实施方式中，合成子由包括至少一种ds多核苷酸和至少一种ss寡核苷酸的混合物的一组多核苷酸组装。在本发明的方法的实施方式中，合成子由包括在除亚群成员之间变化的子序列以外彼此相同的多核苷酸亚群的一组多核苷酸组装。方法的实施方式可以用于制备各种合成子，包括编码序列、载体、用于基因工程和表达组件的引导分子(guidemolecule)。在组装之前，初始ds多核苷酸的长度可以在100碱基至30kb的范围内，尽管该范围以外的多核苷酸也可以在某些情形中使用。例如，在一些实施方式中，单个片段大小可以至多20kb～30kb或更长，或者短至30碱基～500碱基。而且，在一些实施方式中，不同大小的片段可以在组装反应中接合。在一个实施例中，将长多核苷酸(例如，长度5kb～20kb的片段)与短核苷酸(例如，长度100碱基～500碱基的片段)接合。新组装的合成子可以使用单分子测序方法直接进行测序，或者在克隆或扩增之后进行测序。在一个实施方式中，该组成员可以包含长度小于2kb的重叠序列，例如，在15～200个核苷酸的范围内，例如，20～100个核苷酸。在一个实施方式中，提供一种组合物，其中组合物具有5’-3’核酸外切酶；链置换聚合酶；和包含浓度范围7mm～150mm(例如20mm～50mm)的钾盐(比如kcl)的缓冲液。除钾盐以外，还可以使用范围10mm～100mm(比如20mm)的钠盐(例如氯化钠)。组合物中可以包括ss结合蛋白质。在一些实施方式中，组合物不含拥挤剂，比如聚乙二醇(peg)、ficoll或葡聚糖。在一些实施方式中，组合物不含非链置换聚合酶。在另一个实施方式中，用于形成合成子的组合物中包括多核苷酸和/或寡核苷酸片段。在方法的另一个实施方式中，一组寡核苷酸可以在不存在非链置换聚合酶的情况下使用一种组合物来接合，上述组合物包括除ss结合蛋白质以外或者代替ss结合蛋白质的拥挤剂比如聚乙二醇(peg)、ficoll或葡聚糖，和至少7mm钾盐比如kcl，连同链置换聚合酶和5’-3’核酸外切酶。在一个实施方式中，钾盐浓度低于150mm，例如，20mm～50mm。另外提供一种用于多核苷酸组装的试剂盒，其包括：i.5’-3’核酸外切酶；ii.任选的连接酶；iii.链置换聚合酶：和iv.ssdna结合蛋白质。在某些实施方式中，例如，试剂盒还可以包括dntp和/或缓冲剂。试剂盒的组分可以在单独的容器中(例如，一个或多个不同的反应管)，或者，试剂盒的组分可以在单一容器中。组分可以是冻干的，或者是溶液状态的，或者一部分冻干并且一部分为溶液状态。组分可以部分或全部固定化在固体表面比如球珠(bead)上或者反应室表面上，或者是溶液状态的。可以将组分加入到部分或全部固定化的或者溶液状态的靶标多核苷酸中。在一些实施方式中，试剂盒可以包含一种或多种试剂盒组分混合物。在一些实施方式中，试剂盒不包含非链置换聚合酶或拥挤剂。在一个实施方式中，提供与seqid:1的序列同一性为至少80％、85％、90％或95％的聚合酶用于组装混合物。在另一个实施方式中，提供结合结构域与seqid:2的序列同一性为至少80％、85％、90％或95％的聚合酶用于组装组合物。在另一个实施方式中，提供与seqid:3的序列同一性为至少80％、85％、90％或95％的聚合酶用于组装混合物。这些组合物可以在其中聚合酶为链置换性的反应条件下使用。组合物可以在其中与聚合酶活性有关的任何3’核酸外切酶活性呈活性的反应条件下使用。组装反应可以使用ss或ds核酸发生。可以组装任意数目的片段，例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20或更多个片段。可以将化学合成的ss多核苷酸组装到ds重叠片段中，并且与线性化载体的末端杂交并连接，以形成适合于克隆的合成子。另外可选地，可以将双链片段组装到合成子中，并插入到用于克隆或pcr或恒温扩增的载体中(参见，例如，图2a～2c)。通过将核酸片段与线性化ds载体的3’ss末端杂交，还可以将ss核酸片段直接插入到载体中(参见，例如，图7)。ss核酸可以在ss核酸片段与线性化载体的3’ss末端杂交的同时、在其之前或之后通过重叠互补末端来组装。在插入到载体中用于克隆之前，组装的片段可以通过pcr或恒温方法进行扩增。核酸片段可以包含随机化核苷酸序列或简并密码使得能够形成包含在可变区的每个核苷酸位置处的代表性变体的文库。随机序列可以位于每个末端处的给定序列之间。在一个实施方式中，位于指定序列之间的随机序列可以用于与第二核酸片段(例如第二ss基因组多核苷酸)或者线性化载体的末端杂交。在一个实施例中，随机序列与用于将cas9蛋白质引导至用于基因编辑的靶标核酸的靶标基因组序列杂交(参见，例如，图9a～9c)。因此，根据本发明这一方面，该方法可以用于将一组多核苷酸的至少一种成员与第二核酸片段(比如第二ss基因组多核苷酸)或者与线性化载体末端杂交。例如，该方法可以用于将该多核苷酸组的至少一种成员与靶标基因组序列杂交，以在基因编辑方法中将cas核酸内切酶比如cas9引导至靶标基因组核酸。在一些实施方式中，提供一种将多种多核苷酸组装到合成子的方法，其包括：将多种多核苷酸与包括5’-3’核酸外切酶、链置换聚合酶、任选的连接酶、ss结合蛋白质和缓冲剂的组合物合并，其中每种多核苷酸均在一条链上具有3’ss末端多核苷酸序列：其可以与第二多核苷酸上的互补性ss互补序列杂交，并且可以任选地在单一容器中在恒温条件下接合以形成连续的双链多核苷酸。合成子可以进一步在其末端与用于扩增和/或克隆的线性化质粒的末端接合。在一些实施方式中，整个组装方法可以作为“一步”反应(在单一管中，其不必在反应开始之后的期间打开)进行。在一个实施例中，组分在反应容器中混合在一起，并在40℃至60℃的温度下孵育一段时间，例如，5分钟至12小时，从而制备合成子。在一方面，该方法包括在要通过聚合酶接合的多核苷酸中进行链置换的步骤，上述聚合酶与seqidno:1或seqidno:102和/或seqidno:2或seqidno:3的氨基酸序列同一性为至少90％。在方法实施方式中，不要求有额外的3’-5’核酸外切酶回切(chewback)步骤。另一方面还包括通过在反应混合物中包括最小浓度为7mm的钾盐提高组装反应效率，其中钾盐的实例有kcl。在一方面，提供一种方法，其中多核苷酸包含给定的序列末端之间的随机序列。在另一方面，该方法还包括筛选具有与基因组dna杂交的活性的随机序列以及鉴定具有杂交活性的随机序列。在另一方面，该方法还包括通过转录具有杂交活性的随机序列以形成rna，以及在cas核酸内切酶的存在下使用用于基因编辑的rna，从而进行基因编辑。在本发明的组合物、试剂盒或方法的一个实施方式中，本发明的组合物、试剂盒或方法中使用的链置换聚合酶可以是非天然存在的，比如，突变体或融合蛋白。在本发明的组合物、试剂盒或方法中，非天然链置换融合蛋白酶的特征可以在于聚合酶部分的氨基酸组成与seqidno:33至seqidno:55、或seqidno:1中任意者的同一性为至少90％或95％或99％或100％，或者dna结合部分的氨基酸组成与seqidno:56至seqidno:98或seqidno:2中任意者的同一性为至少90％或95％或99％或100％。在一个实施方式中，氨基酸序列与seqidno:102的同一性可以为90％或95％或98％或99％的聚合酶部分，其与选自氨基酸序列与seqidno:56至seqidno:98中任意者的同一性为至少90％或95％或99％或100％的多肽中的异源性dna结合部分融合。概括来讲，在一方面，提供一种制剂，其包括氨基酸序列与seqidno:3的序列同一性为至少90％的组合物。该制剂还可以包括5’-3’核酸外切酶，例如，t5核酸外切酶。该制剂还可以包括ssdna结合蛋白质，例如，etssb、大肠杆菌reca、t7基因2.5产物、噬菌体λredb或rac原噬菌体rect，更具体地，包括热稳定性ss结合蛋白质，比如etssb。该制剂还可以包括连接酶。在一方面，包括组合物的制剂还可以包括ss结合结构域和5’-3’核酸外切酶，其中该制剂不包括拥挤剂和/或非链置换聚合酶。该制剂还可以包括钾盐。在一方面，包括组合物的制剂还可以包括一组中的多种多核苷酸，其中所述组中的至少一种多核苷酸的序列与所述组中另一种多核苷酸重叠；并且其中多核苷酸选自：(i)ds多核苷酸；(ii)ss寡核苷酸；(iii)至少一种ds多核苷酸和至少一种ss寡核苷酸；和(iv)除了在亚群成员之间变化的序列以外，否则彼此相同的多核苷酸亚群。在一方面，多核苷酸组具有至少3种成员或至少4种成员或至少5种成员。通常，用于制备合成子的方法包括：在合适的反应条件下将还包括5’-3’核酸外切酶以及任选的连接酶和ssdna结合蛋白质的权利要求1的组合物与形成一组的多种多核苷酸一起孵育，其中所述组的成员具有重叠序列；以及将至少两种多核苷酸接合，以制备合成子。在一方面，该多核苷酸组包含至少3种成员或至少4种成员或至少5种成员。在一方面，该制剂还包括连接酶。一方面，该制剂还包括ssdna结合蛋白质。在一方面，多核苷酸为ds，并且ds多核苷酸是重叠pcr产物、重叠限制片段或者由ss寡核苷酸组装。在一方面，多核苷酸是ss寡核苷酸。在一方面，多核苷酸组包括至少一种ds多核苷酸和至少一种ss寡核苷酸。概括来讲，本发明提供一种试剂盒，其包括根据权利要求1的制剂和5’-3’核酸外切酶，如例如t5核酸外切酶。在一方面，试剂盒还可以包括ss结合蛋白质。在另一方面，试剂盒可以包括连接酶。在另一方面，试剂盒可以包括缓冲剂。在一方面，试剂盒不包括拥挤剂。在一方面，组合物和5’-3’核酸外切酶在相同容器中。在另一方面，组合物和5’-3’核酸外切酶在不同容器中，任选地是在适合于合并在单一容器中的缓冲剂中。概括来讲，本发明提供一种用于组装合成子的组合物，其包括：5’-3’核酸外切酶，其在一方面具有ss核酸内切酶活性，例如，与seqidno:98的序列同一性为90％；链置换聚合酶，其包括b族链置换聚合酶，其优选为非天然存在的，比如源自天然存在的聚合酶的突变体或融合蛋白，其可以另外地为热稳定性的；任选的ssdna结合蛋白质，例如，ssdna结合蛋白质为etssb、大肠杆菌reca、t7基因2.5产物、噬菌体λredb或rac原噬菌体rect；和非天然存在的缓冲剂，其中组合物不包括拥挤剂和/或非链置换聚合酶。在一方面，组合物还包括连接酶和/或ss结合结构域。在一方面，组合物包括一组至少两种多核苷酸(多种多核苷酸)。在一方面，组合物不包括非链置换聚合酶。在另一方面，组合物不包括9°n、phusion、vent或pfudna聚合酶。在一方面，组合物中的链置换聚合酶是融合蛋白，其中聚合酶部分的氨基酸序列与seqidno:1或seqidno:102或seqidno:33至seqidno:55中任意者的同一性为至少90％。例如，融合蛋白的氨基酸序列与seqidno:1或seqidno:102和seqidno:2的同一性可以为至少90％。例如，链置换聚合酶与seqidno:3的序列同一性可以为至少90％。在一方面，组合物可以包括浓度至少7mm的钾盐。在一方面，组合物可以包括一组多核苷酸，其中所述组中的至少一种多核苷酸具有与所述组中另一种多核苷酸重叠的序列，并且其中多核苷酸选自：(i)ds多核苷酸；(ii)ss寡核苷酸；(iii)至少一种ds多核苷酸和至少一种ss寡核苷酸；和(iv)除了在亚群成员之间变化的序列以外，否则彼此相同的多核苷酸亚群。在一方面，该多核苷酸组中至少一种成员包含位于每个末端处给定序列之间的用于与第二ss基因组多核苷酸杂交的随机序列，其中，例如，随机序列为ss，并且其能够与用于将cas蛋白质引导至用于基因编辑的靶标基因组核酸的靶标基因组序列杂交。概括来讲，本发明提供用于形成合成子的方法，其包括在合适的反应条件下孵育包含一组具有重叠序列的多核苷酸的任意上述组合物；以及将至少一些多核苷酸与其他多核苷酸接合，以制备合成子。在该方法的在一方面，所述组中全部或部分多核苷酸为ds。在另一方面，ds多核苷酸是重叠pcr产物；重叠限制片段或者由互补ss寡核苷酸组装的合成ds分子，其中这些寡核苷酸可以在合成器中制备。在一方面，所述组中的全部或部分多核苷酸是ss寡核苷酸。在一方面，该多核苷酸组包括至少一种ds多核苷酸和至少一种ss寡核苷酸。在一方面，该多核苷酸组包括除了在亚群成员之间变化的序列以外，否则彼此相同的多核苷酸亚群。在一方面，多核苷酸的重叠序列长度小于2千碱基。在该方法的一方面，链置换聚合酶包括与seqidno:1、seqidno:2、seqidno:3、seqidno:33至seqidno:96或seqidno:102中任意者的同一性为至少90％的氨基酸序列。在该方法的一方面，该多核苷酸组中至少一种成员包含给定的序列末端之间的随机序列。该方法的另一方面包括筛选具有与基因组dna杂交的活性的随机序列以及鉴定具有杂交活性的随机序列。该方法的另一方面包括通过转录具有杂交活性的随机序列以形成rna，以及在cas蛋白质的存在下使用用于基因编辑的rna，从而进行基因编辑。概括来讲，本发明提供一种用于多核苷酸组装的试剂盒，其包括：5’-3’核酸外切酶；链置换聚合酶；和任选的ssdna结合蛋白质，其中该试剂盒任选地不包括拥挤剂和/或非链置换聚合酶。在一方面，该试剂盒包括连接酶。在另一方面，该试剂盒包括dntp。在另一方面，该试剂盒包括缓冲剂。在另一方面，该试剂盒的单个组分可以在相同的容器或单独的容器中，比如一个或多个不同的储存或反应容器。概括来讲，本发明提供一种组合物，其包括聚合酶融合蛋白，其中聚合酶融合蛋白包括与seqidno:2、seqidno:56至seqidno:96中任意者的同一性为至少90％的氨基酸序列和异源性聚合酶结构域。在一方面，聚合酶融合蛋白包括与seqidno:2中任意者的同一性为至少90％的氨基酸序列；和异源性聚合酶结构域。概括来讲，本发明提供一种包括聚合酶融合蛋白的组合物，其中聚合酶融合蛋白包括氨基酸序列与seqidno:1、seqidno:33至seqidno:55或seqidno:102中任意者的同一性为至少90％的聚合酶结构域；和异源性dna结合结构域。在一方面，聚合酶融合蛋白包括氨基酸序列与seqidno:1的同一性为至少90％的聚合酶结构域；和异源性dna结合结构域。在一方面，聚合酶融合蛋白具有氨基酸序列与seqidno:102的同一性为至少90％的聚合酶结构域；和异源性dna结合结构域。在一方面，聚合酶融合蛋白的氨基酸序列与seqidno:3的同一性为至少90％。在一方面，上述组合物还包括5’-3’核酸外切酶，比如t5核酸外切酶。在一方面，组合物还包括单链dna结合蛋白质，例如选自etssb、大肠杆菌reca、t7基因2.5产物、噬菌体λredb或rac原噬菌体rect中的单链结合蛋白质。在一方面，组合物可以包括连接酶。在一方面，连接酶是热稳定性的。在一方面，组合物不包括拥挤剂和/或非链置换聚合酶。在另一方面，组合物还包括dntp。在另一方面，组合物还包括浓度至少7mm的钾盐。组合物的一方面包括一组多核苷酸，其中所述组中的至少一种多核苷酸的序列与所述组中另一种多核苷酸重叠；并且其中多核苷酸选自：(i)ds多核苷酸；(ii)ss寡核苷酸；(iii)至少一种ds多核苷酸和至少一种ss寡核苷酸；和(iv)除了在亚群成员之间变化的序列以外，否则彼此相同的多核苷酸亚群。概括来讲，本发明提供一种用于制备合成子的方法，其包括将一组多核苷酸与特征在于上文所述的包括聚合酶的组合物一起孵育，其中单个多核苷酸包含与其他多核苷酸中的序列重叠的序列，其中不同多核苷酸的重叠序列能够在合适的反应条件下交叉杂交，并且，例如其中重叠区小于2千碱基，其中组合物还包括5’-3’核酸外切酶和任选的连接酶和ssdna结合蛋白质；以及将多核苷酸接合，以制备合成子。在不同的方面，组合物包括连接酶；和/或ssdna结合蛋白质。在另一方面，所述组中一种或多种多核苷酸为ds，其中ds多核苷酸是pcr产物、重叠限制片段或者由ss寡核苷酸组装；和/或一种或多种多核苷酸是ss寡核苷酸；和/或该多核苷酸组包括至少一种ds多核苷酸和至少一种ss寡核苷酸。概括来讲，本发明提供一种用于多核苷酸组装的试剂盒，其包括上述聚合酶融合蛋白，和5’-3’核酸外切酶；和ssdna结合蛋白质。在一方面，该试剂盒可以包括任意或全部的连接酶、dntp和缓冲剂，其中试剂盒组分可以在相同容器中或不同容器中。附图说明技术人员将会理解到，下述附图仅仅出于说明目的。附图无意以任何方式对本发明教导的范围加以限定。图1a～1e示出实施例1所述的试验如何区分链置换和非链置换聚合酶。该试验确认，t4dna聚合酶是非链置换的，并在封闭寡核苷酸(blockingoligonucleotide)(2)处在44个核苷酸长度时终止模板dna的合成(图1c)，而bst聚合酶(图1d)和非天然聚合酶(图1e)是链置换的，并可以通过置换封闭寡核苷酸(长度27个核苷酸)继续fam引物(1)的dna合成。图1a示出以下酶试验中使用的dna模板上引物(1)和封闭寡核苷酸(2)的序列和位置。图1b示出毛细管电泳之后得到的样品中观察到的荧光，其中不加入酶，并且起始原料在与24个核苷酸的fam引物对应的位置处形成峰。图1c示出加入t4dna聚合酶的结果。引物延长至最终长度44个核苷酸(24个核苷酸加上20个核苷酸)，但由封闭寡核苷酸终止。图1d示出，bstdna聚合酶(大的片段)对封闭寡核苷酸进行链置换，并通过将引物延长至总长度72个核苷酸(24+20+27+da)对模板进行拷贝。图1e示出作为校对聚合酶的b族链置换dna聚合酶对封闭寡核苷酸进行链置换，并通过将引物延长71个核苷酸(24+20+27)对模板进行拷贝。图2a～2c示出dna组装方法中的步骤。图2a示出将5种片段中每一个的扩增子加入到具有氨苄青霉素抗性标志物的5种质粒中。用产生具有重叠区以及两侧为noti限制性位点的扩增子的引物对5种片段进行初始扩增。noti切割产生粘性末端。noti限制性位点(3)允许由载体释放出每种扩增子。限制性酶切片段与相邻片段具有80个碱基对重叠区(4)。在图2c中，出于方便及降低成本，第一片段和相邻的试剂载体末端以及最后片段和相邻的试剂载体末端之间的重叠为15～25核苷酸，例如，20核苷酸，但这并不意在是限制性的。图2b示出noti切割的任选测序的片段(5)(由载体回收到的扩增子)，然后将其在单个反应容器中用包括t5/5’-3’核酸外切酶、具有3’-5’核酸外切酶活性的dna聚合酶、ss结合蛋白质etssb(newenglandbiolabs,ipswich,ma)和dna连接酶的酶混合物处理(6)～(8)。尽管在此使用noti，取决于方便性，也可以使用其他的限制性核酸内切酶以用于切割。可以用两种或更多种限制性核酸内切酶进行双消化。例如，已经发现，用两种限制性核酸内切酶对载体dna进行双消化降低来自未切割载体的本底。将重叠ssdna序列与相邻片段杂交。t5核酸外切酶在每个片段上将dna链从5’到3’回切(chewback)，以暴露3’ss区(6)，这促使片段在ss结合蛋白质的存在下一起退火(7)。通过与链置换聚合酶相关的3’-5’核酸外切酶活性的手段，达到2个碱基翼(flap)的除去，然后通过链置换聚合酶进行延长，以充填组装产物中的间隙(8)。任何残余的切口或5’翼可以通过连接酶和/或t5核酸外切酶修复。图2c示出现在按顺序接合并插入到用于转化到细菌宿主中的携带氯霉素抗性基因(cam)的第二质粒中的5个片段(片段(frag)1至片段5)。图3a和3b示出，使用氯霉素板以选择在板上生长的菌落，并且在iptg和x-gal的存在下，包含laclz基因的那些菌落产生蓝色菌落。试验提供了对其中基因以功能性形式有效组装的克隆的定量评价。图3a示出仅有氯霉素。图3b示出氯霉素+iptg+xgal。图4示出质粒事实上确实包含整个基因。对图2b所示的组装产物进行pcr扩增，以确认所有片段在转化之前得以接合和连接。条带1和2是重复的pcr结果。条带m是来自newenglandbiolabs,ipswich,ma的2-logdna梯状带(ladder)。图5示出通过菌落数目测定的组装混合物中kcl的影响。缓冲液中使用的kcl浓度增加，表现出组装混合物中使用链置换聚合酶的组装的准确性/效率增加。左边的柱状图(t26)不含kcl，而右边的柱状图(t26k)包含25mmkcl，其示出效率提高1.5倍。无论组装条件如何，这一提高均发生。如果在不存在ss结合蛋白质的情况下使用peg或其他拥挤剂，预计有类似的相对效率提高。图6示出根据制造商所提供的方案，实施例2中所述的混合物(链置换聚合酶/ss结合蛋白质/5’-3’核酸外切酶/连接酶)(mix1)和市售gibsonmix(gamm)(非链置换聚合酶和聚乙二醇)(syntheticgenomics,lajolla,ca/newenglandbiolabs,ipswich,ma)之间的比较。mix1产生显著更高的dna组装和转化效率。图7示出ssdna寡核苷酸和dsdna片段之间的dna组装的概略图。将ss靶标dna寡核苷酸插入到dna载体中。已合成出ss靶标dna寡核苷酸，以在每个末端上具有20～30个核苷酸的与3’载体末端的重叠区。然而，该寡核苷酸的尺寸可以具有小于20个核苷酸的重叠区，例如小于15个核苷酸或小于10个核苷酸，或者另外可选地大于30个核苷酸，例如，至少40个或50个或60个核苷酸或更多。在重叠区之外，该寡核苷酸优选地具有位于末端之间的不重叠的1个或更多个核苷酸。将包含5’-3’核酸外切酶、链置换聚合酶、连接酶和ss结合蛋白质的组装主混合物加入到ss寡核苷酸和载体的混合物中，以使得dsdna载体的5’末端得以回切(chewedback)，以产生ss突出端(9)。然后ssdna的3’末端能够退火至载体的5’末端，然后dna聚合酶对ss模板进行复制，以填充空隙，并产生平端dsdna。将切口通过连接酶密封(10)。再次，核酸外切酶(在此为t5核酸外切酶)这次在靶标dna的平端回切5’末端，产生3’ss区，以使得互补序列退火，并完成靶标dnads整合到dna载体中(11)。片段可以借助填充空隙的dna聚合酶和密封切口的连接酶退火(12)以制备合成子。图8a～8c示出由短ss寡核苷酸桥接dsdna的工作流程。(方案描述于图7中)。图8a提供用于整合到在此示出为具有ofp报告子的crispr核酸酶载体的dsdna载体中的短ss寡核苷酸序列的实例。图8b示出的工作流程起始于ss寡核苷酸和dscrispr核酸酶载体(9424bp)，其用5’-3’核酸外切酶、链置换聚合酶、连接酶和ss结合蛋白质处理(13)，以产生完整的ds环状dna。将该dna转化到感受态细胞中(14)。过夜孵育之后，通过微制备对菌落进行分析，然后进行质粒测序(15)。图8c示出插入的序列和相邻序列、u6启动子序列(载体)、设计的ss寡核苷酸(71聚体(mer))的序列和骨架(scaffold)模板特异性序列(载体)。将在每个末端包括25个核苷酸的重叠区(黑体示出的靶标dna的21核苷酸)的ss寡核苷酸(71聚体)适当地整合到宿主细胞中的载体中。图9a～9c示出，与图8中类似的工作流程可以用于在重叠端之间具有简并碱基的ss寡核苷酸。同样，图9a中的起始序列示出于工作流程(图9b)之上，并且来自组装库的菌落的sanger测序结果示出于下方(图9c)，实线是指sgrna靶标序列库。sgrna靶标序列包含21个可变核苷酸位置，提供421种变体。该库包含每种可能的变体，并且每种变体适于克隆反映重叠末端和载体之间序列的简并性。图9a示出包含简并碱基的ss寡核苷酸的序列。将图9a的sgrna靶标序列插入在载体的u6启动子序列和骨架模板特异性序列之间(16)，将其转化到宿主细胞中(17)，并通过微制备和测序进行合成子分析(18)，如上文及在此所述。对来自组装库的克隆进行sanger测序。图9c提供序列的实例。图10示出将组装反应产物转化到大肠杆菌中之后选自一板的187个菌落的结果。对每个菌落进行pcr扩增和测序，以确认ssdna的插入，并对简并碱基的分布进行分析。在此示出的结果确认，不同的菌落确实包含不同的简并序列。未检测到偏性。首先通过将序列转化成fastq文件，然后在github上使用来自fastx工具包的fastx_quality_stats工具，进行分析。使用来自berkeley的weblogo产生序列标识。术语说明除非另外指出，本文使用的所有科技术语的含义与本发明所属领域普通技术人员所通常理解的相同。singleton等人，dictionaryofmicrobiologyandmolecularbiology，第二版，johnwileyandsons,newyork(1994)和hale&markham,theharpercollinsdictionaryofbiology,harperperennial,n.y.(1991)为技术人员提供了许多本文所用术语的通常含义。为清楚和便于参考起见，以下仍对某些术语加以定义。如本文所用，如基因合成领域所用的术语“合成子”是指多核苷酸组装体。多核苷酸组装可以包括组装一定大小的重叠片段，其可以在寡核苷酸合成器上制备，对于每个合成的多核苷酸，目前通常为2000～3000个碱基。另外可选地，重叠片段可以由连接有接头(adaptor)以提供重叠序列的天然存在的核酸通过pcr获得。出于组装目的，对每个片段的大小没有限定。依赖于末端处的重叠序列，可以将许多片段端到端地组装，使得能够准确、有效地产生具有任意所需长度的构建体。优选地，合成子是由较短的多核苷酸的组装体形成的不含间隙和切口的较长的连续多核苷酸。然而，由核酸片段组装体产生的合成子的长度不限定于任何具体的大小。如本文所用，术语“5’-3’核酸外切酶”是指从5’末端，即以5’至3’方向降解dna的核酸外切酶。所关注的5’-3’核酸外切酶可以在平端处以及在某些实施方式中在3’和/或5’突出端处从dsdna链的5’末端除去核苷酸。t5核酸外切酶、λ核酸外切酶和t7核酸外切酶均为5’-3’核酸外切酶的实例。在某些实施方式中，优选t5核酸外切酶。t5核酸外切酶额外地具有ss核酸内切酶活性。如本文所用，术语“连接酶”是指可以具体地在切口处将dna分子的3’末端与另一dna分子的5’末端共价接合的酶。尽管已知有许多其他的连接酶并可以在本文使用，连接酶的实例包括t7连接酶、t4dna连接酶、大肠杆菌dna连接酶和taq连接酶。如本文所用，术语“链置换聚合酶”是指能够置换酶下游的一个或多个核苷酸比如至少10个或100个或更多个核苷酸的聚合酶。链置换聚合酶可以与phusion区分，其中phusion在本领域公认的定义是非链置换聚合酶。在一些实施方式中，在至少50℃或至少55℃的温度下，链置换聚合酶是稳定、有活性的(包括链置换活性)。taq聚合酶是切口翻译聚合酶，因此，其不是链置换聚合酶。如本文所用，术语“单链(ss)dna结合蛋白质”是指与ssdna结合、防止早熟退火、保护ssdna不被核酸酶和聚合酶消化、和/或去除dna二级结构以使其他酶对其有效发挥功能的蛋白质。优选在本文所述的组合物中包括ss结合蛋白质，以优化合成子形成的效率。尽管已知有许多其他者，并可以在本文使用，例如λ噬菌体redb、rac原噬菌体的rect和下文所列的序列，ssdna结合蛋白质的实例有t4基因32蛋白质、大肠杆菌ssb、t7gp2.5ssb、和噬菌体phi29ssb、以及etssb。在一些情形中可以使用在50℃下稳定的热稳定性ssdna结合蛋白质。因此，在本发明的组合物、试剂盒或方法的一个实施方式中，ssdna结合蛋白质为t4基因32蛋白质、大肠杆菌ssb、t7gp2.5ssb、噬菌体phi29ssb、etssb、λ噬菌体redb或rac原噬菌体rect。在一个实施方式中，ssdna结合蛋白质是etssb。在本发明的组合物、试剂盒或方法的一个实施方式中，ssdna结合蛋白质是热稳定性的(即，在40℃～60℃下稳定)。如本文所用，术语“缓冲剂”是指当向溶液中加入酸或碱时使得溶液抵抗ph改变的试剂。本发明的组合物、试剂盒和方法中可以使用的合适的非天然存在的缓冲剂的实例包括，例如，tris、hepes、taps、mops、两性离子缓冲剂(tricine)或mes。术语“非天然存在的”是指组合物在自然界中不存在。本文所述的任何蛋白质均可以是非天然存在的，其中术语“非天然存在的”是指蛋白质具有与其天然状态下不同的氨基酸序列和/或翻译后修饰方式。例如，非天然存在的蛋白质可以在蛋白质的n-末端、c-末端和/或在n-末端和c-末端之间具有一个或多个氨基酸替换、缺失或插入。“非天然存在的”蛋白质的氨基酸序列可以与天然存在的氨基酸序列不同(即，与天然存在的蛋白质的氨基酸序列的同一性小于100％)，但与天然存在的氨基酸序列的同一性为至少80％、至少85％、至少90％、至少95％、至少97％、至少98％或至少99％。在某些情形中，如果由不同的(例如，细菌)细胞制备非天然存在的蛋白质，其可以包含n-末端甲硫氨酸或者可以缺少一个或多个翻译后修饰(例如，糖基化、磷酸化等)。“突变的”蛋白质相对于野生型蛋白质可以具有一个或多个氨基酸替换，并且可以包括“融合”蛋白。术语“融合蛋白”是指由多种在天然状态下未接合的多肽组分组成的蛋白质。融合蛋白可以是2个、3个或甚至4个或更多个不同蛋白质的组合。术语多肽包括融合蛋白，包括，但不限于，两个或更多个异源性氨基酸序列的融合，多肽与以下的融合：异源性靶标序列、连接序列(linker)、免疫学标签、可检测的融合配偶体比如荧光蛋白、β-半乳糖苷酶、荧光素酶等，等等。融合蛋白可以具有一个或多个加在蛋白质n-末端、c-末端和/或中间部分的异源性结构域。如果融合蛋白的两部分是“异源性”的，则它们在其天然状态下不是相同蛋白质的一部分。在上下文述及核酸时，术语“非天然存在的”是指核酸包含：a)与天然状态下的核酸不同的核苷酸序列(即，与天然存在的核酸序列的序列同一性小于100％)，b)一个或多个非天然存在的核苷酸单体(其可以产生不是g、a、t或c的非天然骨架或糖)，和/或c)可以在核酸的5’-末端、3’-末端和/或在5’-和3’-末端之间包含一个或多个其他修饰(即，加入的标记或其他部分)。在上下文述及制剂时，术语“非天然存在的”是指：a)例如由于组分在不同的位置、在不同的细胞中或在不同的细胞区室中而非通过自然组合，这样组分的组合物；b)其相对浓度在自然界未发现的组分的组合物；c)缺少某些通常在自然界中与组分之一关联的组分的组合物；d)自然界中未发现的形式例如干燥、冻干、结晶或含水形式的组合物；和/或e)包含自然界中未发现的组分的组合物。例如，制剂可以包含自然界中未发现的“非天然存在的”缓冲剂(例如，tris、hepes、taps、mops、两性离子缓冲剂或mes)、洗涤剂、染料、反应促进剂或抑制剂、氧化剂、还原剂、溶剂或防腐剂。可能需要使用具有3’核酸外切酶活性的链置换聚合酶。尽管无意拘泥于理论，但需要3’核酸外切酶，以除去双链3’末端上的侧翼序列(flapsequence)，其中侧翼序列可以是酶切的产物，以便从其置于的质粒中提取靶标多核苷酸。这是如实施例中所述的使用noti的情形。然而，如果使用在切除的片段上产生平端的限制性核酸内切酶，可以不要求3’核酸外切酶活性。3’核酸外切酶活性可以通过使用标准dna模板和引物加以常规测定，其中引物具有或不具有非杂交3’核苷酸。如果聚合酶具有3’核酸外切酶活性，则使用任一引物对将会检测到扩增子。如果聚合酶缺少3’核酸外切酶活性，则使用具有非杂交3’核苷酸的引物将检测不到扩增子。如本文所用，术语“钾盐”是指包括但不限于kcl的钾盐。术语“钠盐”是指包括但不限于nacl的钠盐。如本文所用，术语“多核苷酸”包括寡核苷酸，并且其是指任意长度的核酸。多核苷酸可以是dna或rna。除非指定，多核苷酸可以为ss或ds。多核苷酸可以是合成的，例如，在dna合成器中合成，或者是天然存在的，例如，提取自天然的来源，或者源自克隆或扩增的材料。本文所指的多核苷酸可以包含修饰的碱基。如本文所用，术语“多核苷酸组”或“一组多核苷酸”是指至少两种多核苷酸的集合。在一些实施方式中，一组多核苷酸可以包括至少5种、至少10种、至少12种、或至少15种或更多种多核苷酸。如本文所用，术语“重叠序列”是指在两个多核苷酸中互补的序列，并且其中重叠序列为ss，在一种多核苷酸上，其可以与另一种多核苷酸上另一重叠互补ss区杂交。例如，在一组多核苷酸中，重叠序列可以在至少5、10、15或更多种多核苷酸中互补。重叠序列可以在两个不同分子的3’末端处(例如，两个ss寡核苷酸的3’末端，或者第一ds多核苷酸顶部链的3’末端和第二ds分子底部链的3’末端)或者与其接近(例如，大约5、10、20个核苷酸以内)，其中，如果非重叠序列在3’末端处，则非重叠序列可以使用聚合酶的3’-5’核酸外切酶活性去除。重叠序列的长度可以变化，在一些情形中，长度可以为至少12个核苷酸(例如，长度至少15个、20个或更多个核苷酸)，和/或长度可以至多100个核苷酸(例如，长度至多50个、至多30个、至多20个或至多15个核苷酸)。另外可选地，多核苷酸组中的重叠序列可以为2kb或更小，或者1kb或更小，或者小于900碱基、800碱基、700碱基、600碱基、500碱基、400碱基、300碱基、200碱基或100碱基。优选地，重叠序列长度在15个核苷酸至80个核苷酸范围内，例如，至多20个、至多25个、至多30个、至多35个、至多40个、至多45个、至多50个、至多55个、至多60个、至多65个、至多70个、至多75个或至多80个核苷酸。重叠的最小长度可以通过tm定义，其优选地等于或大于48℃。如本文所用，术语“多核苷酸组装”是指2个或更多个、4个或更多个、6个或更多个、8个或更多个、10个或更多个、12或更多个、15或更多个多核苷酸，例如4个或更多个多核苷酸彼此接合产生更长的多核苷酸的反应。在许多实施方式中，多核苷酸组装反应的产物，即“组装的多核苷酸”或“合成子”应当包含一个拷贝的每种重叠序列。如本文所用，术语“在合适的反应条件下孵育”是指保持反应在合适的温度和时间，以实现所需的结果，即多核苷酸组装。适合用于本发明方法的酶和试剂的反应条件是已知的(例如，如本文实施例中所述)，因此，本发明方法的合适的反应条件可以很容易确定。这些反应条件可以根据所用的酶而改变(例如，取决于其最佳温度等)。如本文所用，术语“等温”是指对于组装进行不要求主动调节温度的温度条件。水浴或加热块温度无关紧要的变化在术语等温的含义范围之内。例如，术语“等温”可以指反应开始之后不要求热变性步骤的反应条件。更具体地，等温方法不涉及热循环，即在高于90℃的变性温度和退火/延长温度之间的循环。等温条件通常涉及在低于90℃的温度下孵育一段时间(例如，5分钟至12小时或更长时间)。在一个实施方式中，等温扩增反应在30℃～75℃，例如40℃～60℃范围内的温度下进行。如本文所用，术语“接合”是指在两个序列之间产生共价连接。如本文所用，术语“组合物”是指除了列出的那些之外，可以包含其它试剂例如甘油、盐、dntp等的试剂的组合。组合物可以是任意形式的，例如，含水的或冻干的，并且可以是任意状态的(例如，冷冻的或液体形式)。如本文所用，“载体”是片段或合成子可以整合到其中使得可以在宿主细胞中复制改造载体的合适的dna。线性化载体可以通过环状载体的限制性核酸内切酶消化或者通过pcr产生。片段和/或线性化载体的浓度可以通过凝胶电泳或其他方式测定。本文使用的任何一种或多种蛋白质(例如，连接酶、ssbp、5’-3’核酸外切酶或聚合酶等)可以是温度敏感的或热稳定的，其中，如本文所用，术语“温度敏感”是指酶在65℃的温度下10分钟之后丧失其活性的至少95％，并且术语“热稳定”是指酶在65℃的温度下10分钟之后保持其活性的至少95％。具体实施方式在对各种实施方式进行更详细的说明之前，应当理解到，本公开的教导不限于所述的具体实施方式，因此，其当然可以变化。还应当理解到，本文所用的术语仅出于说明具体实施方式的目的，而无意于进行限定，因为本发明教导的范围将仅由其权利要求书进行限定。尽管本发明的教导结合各种实施方式进行说明，但无意于将本发明的教导限制于这些实施方式。相反，如本领域技术人员将会理解到，本发明的教导包括各种替代方式、修改方式和等同方式。在提供数值范围时，应当理解到，位于该范围上限和下限之间的每个中间值(每个中间值精确到下限的单位的十分之一，除非上下文中有清楚地相反表示)，以及任何其他所述的或在其所述范围内的中间值均包括在本公开以内。尽管在实施或测试本发明教导内容时也可以使用与本文所述的那些类似或等同的任何方法和材料，但是现对一些示例性方法和材料加以说明。任何出版物的引用是出于其公开早于申请日，不应当理解成认可本发明的权利要求由于在先发明而无权早于这些出版物。而且，所提供出版物的日期可能与实际的公开日期不同，其需要独立地确认。必须注意到，如本文和其权利要求中所用的，单数形式“一个”、“一种”和“该”包括复数的指示物，除非上下文中有清楚地相反表示。还注意到，权利要求可以撰写成排除任何任选的要素。这样，该声明意在用作结合权利要求要素的引用使用这些排除性术语如“仅仅”、“仅有”等、或者使用“否定”限定的前提基础。如阅读本公开时对本领域技术人员将会显而易见的是，本文说明和例示的每个单独的实施方式均具有独立的组分和特征，其可以在不脱离本发明教导的范围或精神的前提下很容易地与任何其他若干实施方式的特征分开或组合。任何所引用的方法均可以以所引用事件的顺序或者以任何其他逻辑上可能的顺序实施。在多核苷酸组中重叠的序列可以是任何合适的长度，比如，2kb或更小，或者1kb或更小，或者小于900碱基、800碱基、700碱基、600碱基、500碱基、400碱基、300碱基、200碱基或100碱基。重叠区可以少至8个核苷酸。优选地，重叠序列长度在15个核苷酸～80个核苷酸范围内，例如，至多20、至多25、至多30、至多35、至多40、至多45、至多50、至多55、至多60、至多65、至多70、至多75或者至多80个核苷酸。例如，最小重叠长度可以由tm定义，其优选等于或大于48℃。合成的寡核苷酸和多核苷酸在其用于合成子组装之前可以包含其合成过程中产生的错误。为了在组装之前校正这些错误，期望进行错配修复步骤。就此而言，已经说明了多种在组装之前实现合成核酸的错配修复的方法。合成核酸的种群可以具有随机错误，使得制剂的变性和复性可以显示错配。已经从自然界分离出的蛋白质，比如muthls、cel-1核酸酶、t7endo1、uvrd、t4endovii、大肠杆菌endov(参见us7,851,192和us8,048,664)，可以选择性地与包含错配的dna双链结合；在错配碱基处切割核酸；并任选地基于模板核苷酸序列用正确碱基替代。尽管本领域中有教导非链置换聚合酶必须与ss结合蛋白质、5’-3’核酸外切酶和连接酶一起使用以组装dna片段，本文出人意料地显示，链置换聚合酶可以在发生链置换的条件下使用，并且在起始多核苷酸片段出人意料的低浓度下有效，以有效地由多个片段产生单个核酸。可以用于本发明的组装混合物、组合物、试剂盒或方法的实施方式中的链置换聚合酶的实例包括b族聚合酶成员，比如(但不限于)表1中所确认的任意者(seqidno:33至seqidno:55)。此外，可以使用这些聚合酶的融合，例如，在多种聚合酶和/或ss结合结构域(比如，如表2所示)(seqidno:56至seqidno:97)之间的融合。在实施方式中，表1中的任何聚合酶部分或者与表1中任意这些蛋白质部分的氨基酸序列同一性为至少80％、85％、90％、95％、98％、99％或100％的蛋白质均可以在n-末端处或c-末端处与表2中所述的任何dna结合结构域或者与表2中任意dna结合部分的氨基酸序列同一性为至少80％、85％、90％、95％、98％、99％或100％的蛋白质的部分来融合，以形成本文使用的链置换融合聚合酶。dna结合结构域可以任选地融合在聚合酶的n-末端或c-末端。也可以使用如通过本文提供的试验(参见，例如图1a～1e和实施例1)确定显示为链置换性的其他聚合酶变体或新的分离物。这些来源发现的聚合酶的序列通过genbank很容易获得。由于链置换序列的高度保守性，任何与这些野生型聚合酶的氨基酸序列同一性为80％、85％、90％或95％的变体可以预计具有链置换特性，其可以在预选择的缓冲剂中在实施例1提供的试验中快速并容易地验证，而无需过度实验。在一个实施方式中，本发明的反应混合物、组合物、试剂盒或方法包括或使用与seqidno:1或seqidno:102的序列同一性为至少80％、85％、90％、95％、98％、99％或100％(例如，与seqidno:1或seqidno:102的序列同一性为100％)的链置换聚合酶。在另一个实施方式中，本发明的反应混合物、组合物、试剂盒或方法包括或使用结合结构域与seqidno:2的序列同一性为至少80％、85％、90％、95％、98％、99％或100％(例如，与seqidno:2的序列同一性为100％)的聚合酶。在另一个实施方式中，本发明的反应混合物、组合物、试剂盒或方法包括或使用与seqidno:1或seqidno:102并且与seqidno:2或seqidno:3或seqidno:33至seqidno:97中任意者的序列同一性为至少80％、85％、90％、95％、98％、99％或100％(例如，与seqidno:1或seqidno:102并且与seqidno:2或seqidno:3或seqidno:33至seqidno:97中任意者的序列同一性为100％)的聚合酶。这些组合物可以在其中聚合酶为链置换性的反应条件下使用。组合物可以在其中任何与聚合酶活性有关的3’-5’核酸外切酶活性是有活性的反应条件下使用。当在反应中使用限制性酶比如noti时这一点会有帮助。在该情形中，3’-5’核酸外切酶可以除去双链3’-末端上的侧翼序列。然而，如果使用在切除的片段上产生平端的限制性核酸内切酶，则可以不要求有3’-5’核酸外切酶活性。组装反应可以在等温条件下进行。在一个实施方式中，等温条件为50℃。表1.聚合酶列表methanocaldococcusvulcaniusm7sp-13gi|502573182seqidno:33archaeoglobusfulgidusdsm4304sp-16gi|499180464seqidno:34archaeoglobusprofundusdsm5631sp-17gi|502704426seqidno:35caldicellulosiruptorhydrothermalis108sp-19gi|503168530seqidno:36desulfurococcusmucosusdsm2162sp-27gi|503328138seqidno:37pyrolobusfumariisp-29gi|503791850seqidno:38pyrobaculumoguniensechsp-30gi|379003208seqidno:39staphylothermusmarinusf1sp-33gi|500164563seqidno:40pyrococcusyayaosiich1sp-42gi|503672202seqidno:41thermococcussp.am4-delsp-43gi|503888003seqidno:42thermococcushydrothermalissp-44gi|17375628seqidno:43thermococcusthioreducenssp-45gi|117958105seqidno:44thermococcuswaiotapuensissp-46gi|378813034seqidno:45thermococcussibiricusmm739sp-47gi|506329477seqidno:46pyrococcusglycovoranssp-48gi|7288074seqidno:47pyrococcussp.na2sp-49gi|503513858seqidno:48ferroglobusplacidusdsm10642sp-61gi|502730992seqidno:49palaeococcusferrophilusdsm13482sp-5gi|851288004seqidno:50thermococcusgammatoleransej3sp-50gi|506339349seqidno:51thermococcuscelericrescenssp-51gi|332308985seqidno:52vulcanisaetadistributadsm14429sp-60gi|503101260seqidno:53methanopyruskandleriav19sp-7gi|20094475seqidno:54thermoproteusneutrophilusv24stasp-9gi|171185774seqidno:55表2.dna结合蛋白质在本发明的实施方式中，反应混合物、组合物、试剂盒或方法可以包括或使用5’-3’核酸外切酶，比如t5/5’-3’核酸外切酶，其对温度敏感，并可以通过将温度升高到50℃以上失活。在一个实施方式中，5’-3’核酸外切酶具有核酸外切酶活性以及ss核酸内切酶活性。在一些实施方式中，反应混合物还可以包括连接酶，例如要求nad+的连接酶和/或热稳定性连接酶，例如taq连接酶。在优选的实施方式中，反应混合物可以包括ss结合蛋白质。ss结合蛋白质可以是热稳定性的，例如，etssb。组装反应可以在等温条件下进行。在某些实施方式中，任选使用连接酶。例如，当将组装的片段直接导入到载体中用于宿主细胞转化时，不需要有连接酶，因为宿主细胞比如大肠杆菌能够在体内修复切口。然而，如果出于在转化之前确认正确组装的目的对组装的片段进行扩增，则期望使用连接酶来封闭切口，并且使聚合酶能够扩增整个靶标dna。单个片段的克隆可以使用其序列得自任何数据库或出版物的化学合成的多核苷酸片段，其中多核苷酸片段具有重叠序列。通过将多核苷酸插入到质粒中邻近适用于切割所插入多核苷酸的限制性内切酶位点的位点中，可以在质粒中克隆这些片段。可以使用任何质粒。本实施例使用包含氯霉素基因作为选择标志物的市售pacyc184。可以使用任何选择标志物代替氯霉素抗性基因。类似地，可以选择任何切割酶的特异性识别位点，只要该切割酶能够在寡核苷酸末端特异性切割以产生交错末端或平端，其中除了邻近所关注的片段的末端的工程化的位置以外，特异性切割位点不发生在所关注的片段中。在本实施例中，已经通过dna合成的方法将产生交错末端的8碱基切割者noti的识别位点(cgccggcg)引入邻近于所关注的多核苷酸。但是，该位点可以存在于所选择的质粒中，或者通过扩增引物加在所关注的合成寡核苷酸上。特异性切割酶的实例包括限制性核酸内切酶和归巢(homing)核酸内切酶。一旦所关注的寡核苷酸或dna片段已经化学合成或由现有dna克隆或扩增并克隆到具有选择标志物的载体中，则优选通过酶切割将其切除。然后将已经合成或扩增以便包括与其要接合的相邻片段或寡核苷酸的重叠序列的片段或寡核苷酸在组装反应中组装。在所选择的杂交条件下，反应混合物中的5’-3’核酸外切酶(例如，浓度范围0.004-0.016u/μl)回切(chewback)片段或寡核苷酸5’末端处的任意ss区，并继续回切经过重叠序列区，并可以进一步继续有限的距离(例如，至少100碱基)，以提供3’ss区(参见，例如，图2a-2c和图7)。同时，通过图1a-1e和实施例1的试验确定的链置换聚合酶(例如，浓度范围0.005u/μl-0.5u/μl)修复杂交的ds区和任何残余ss区之间残留的间隙。由于聚合酶是链置换性的，其可以置换额外的下游序列，以形成ss侧翼。但是，t5核酸外切酶的ss核酸内切酶活性将会除去该侧翼，并且任何相关的切口均可以通过连接酶(例如，浓度范围0.001u/μl-20u/μl)修复。片段一经组装到更大一条dna中，将其在选择性压力下在宿主细胞菌落中克隆，来自这些菌落的dna可以从载体中释放，并再次与其他片段组装，并转化到宿主细胞中，从而多次延长dna的大小。宿主细胞可以是感受态细菌细胞，或者可以是酵母细胞或其它真核细胞。本文所述的组装方法经发现非常有效。例如，可以使用0.02nm-100nm寡核苷酸(ss)或dna片段(ds)来组装更大的片段，其中反应中使用的ss寡核苷酸的浓度可以比类似组装反应中使用的dsdna片段的量高至多大约50倍。类似地，可以使用等摩尔浓度的包含单一片段和选择标志物的质粒和类似量的包含组装片段的具有不同选择标志物的载体。这些量意在用于指导，但无论组装效率是否提高，这些量均可以降低。例如，如通过使用lac1z作为指示物的组装体的菌落数目所测定，加入钾盐kcl能够将产生组装体的效率增加1.5倍(参见，例如，图5)。用于两个dsdna分子之间或者导入线性化载体的ss靶标寡核苷酸的组装过程也是非常高效的。本文提供了一个无意于限定的实例，其使用特定/随机序列来识别可引入到细胞中以确定表型改变的crispr-cas基因编辑方案的向导rna。最初，什么序列可以适合于实现该目的可能并不知晓。包含简并序列的文库的产生使得该类型的分析成为可能。基于crispr/cas-9的基因编辑在基因组编辑领域迅速普及。由于最常用的包含cas9的质粒的尺寸，将sgrna或sgrna文库构建至cas9/sgrna表达载体中会很繁琐。使用ssdna寡核苷酸，该方法解决了这一问题。在单独的实施方式中，可以将表2的任何dna结合结构域与bst聚合酶、bst大片段或其突变体融合(参见，例如，us8,993,298和us2015/0152396，其包括本文描述并要求保护的所有bst变体)。试剂盒本公开另外提供用于实施上述方法的试剂盒。在某些实施方式中，试剂盒可以包含：i.5’-3’核酸外切酶，ii.任选的连接酶，iii.链置换聚合酶：和iv.ssdna结合蛋白质。试剂盒组分可以合并在一容器中，或者每个组分可以在其各自的容器中。例如，试剂盒组分可以合并在单个反应管中、或者一个或多个不同的反应管中。上文描述了该试剂盒组分进一步的细节。根据如何实施所述方法，试剂盒还可以包含上文和下文中所述的可以用于该方法的其他试剂，例如，错配修复酶，比如muthls、cel-1核酸酶、t7endo1、uvrd、t4endovii、大肠杆菌endov、缓冲剂、dntp、向其中插入合成子的质粒和/或接收质粒的感受态细胞、对照物等。在一些实施方式中，试剂盒不含非链置换聚合酶和/或拥挤剂。除上述组分以外，试剂盒还包括使用试剂盒组分以实施本方法的说明书。用于实施本方法的说明书通常记录在合适的记录介质上。例如，说明书可以打印在比如纸或塑料等的介质上。这样，说明书可以作为包装说明书存在于试剂盒中、在试剂盒或其组分的容器标签中(即，与包装或分包装关联)等。在其他实施方式中，说明书作为电子存储数据文件存在，存在于合适的计算机可读存储介质中，例如，cd-rom、软盘等。在又一其他的实施方式中，实际的说明书不存在于试剂盒中，而是提供用于从远程资源例如经由互联网获得的方法。该实施方式的实例为包括可以浏览说明书和/或可以从其下载说明书的网络地址的试剂盒。就说明书而言，获取说明书的这一方法记录在合适的基质上。本文所述的用于组装片段并形成合成子的组合物、试剂盒和方法产生ds完全密封dna的产物，其可以用作pcr、rca的模板、或许多种其他分子生物学应用，包括直接转化感受态细菌或转染真核宿主细胞。为了进一步说明本发明，提供以下具体实施例，应理解到其提供是为了对本发明进行说明，而不应理解成以任何方式限定其范围。本文引用的所有参考文献包括2014年8月27日提交的美国临时申请第62/042,527号、2015年7月7日提交的第62/189,599号加上2015年7月16日提交的第62/193,168号，将其并入以供参考。实施例实施例1：确定聚合酶链置换特性的试验开发一种区分链置换和非链置换聚合酶的试验——制备包含10nmfam-引物/模板/封闭寡核苷酸、缓冲液(newenglandbiolabs,ipswich,ma)(图1a)和0.1mmdntp的10μl反应物。图1b是作为对照的不存在任何聚合酶的fam标记引物。将链置换dna聚合酶加入到反应物中，并在50℃下与稀释10倍的1μl样品一起孵育30分钟，并通过毛细管电泳进行分析，fam引物通过被置换的封闭寡核苷酸得以延长。结果示出于图1d-1e中。图2d中确定的链置换聚合酶bst聚合酶的峰位置与非天然聚合酶spb49f观察到的峰对应。少量迁移的原因在于3’-5’核酸外切酶活性引起的spb49f产生的平端，这在bst聚合酶中并不存在，使得bst聚合酶复制的产物具有3’da。图1c示出了非链置换聚合酶-t4dna聚合酶的产物，其中合成在封闭引物处终止。实施例2：使用链置换聚合酶由6片段合成大的dna分子并使用链置换聚合酶确认组装有效使用pcr克隆试剂盒(newenglandbiolabs,ipswich,ma)分别由pcr产物(片段(frags)1、2、3、4、5，其一起覆盖lacl-和lacz基因区)构建质粒a、b、c、d和e。在该实验中，使用以下浓度的整合到单独质粒中的5个不同片段：50ng的各pcr(“片段”来源)，和25ng的包含氨苄青霉素的质粒pminittm载体(neb#e1202)。首先使用pcr对5种用于组装的片段进行扩增。制备lacl-laczdna片段组装系统中使用的引物如下：5placiz-pacyc184vf1ttggtctggtgtcaaaaatgaatcgtcacggcgatttatg(seqidno：4)5placiz-pacyc184vr1gggtcattttcggcgaggactgcatcaacgcatatagcg(seqidno：5)not-izf1gcggccgcgtcctcgccgaaaatgacccagag(seqidno：6)not-izr1gcggccgctggtgtcgatggtagaacgaagcg(seqidno：7)not-izf2gcggccgccccactgacgcgttgcgcgagaag(seqidno：8)not-izr2gcggccgcggctgcgcaactgttgggaagggc(seqidno：9)not-izf3gcggccgctgcagcacatccccctttcgccag(seqidno：10)not-izr3gcggccgcatgatgctcgtgacggttaacgcc(seqidno：11)not-izf4gcggccgcaggtgcggattgaaaatggtctgc(seqidno：12)not-izr4gcggccgctcaccgcttgccagcggcttacca(seqidno：13)not-izf5gcggccgcgaatacctgttccgtcatagcgat(seqidno：14)not-izr5gcggccgctcatttttgacaccagaccaactgg(seqidno：15)将扩增的片段克隆并测序，以确认扩增过程中没有引入错误。pcr扩增的片段1的序列(seqidno：16)：gcggccgcgtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataaccaacgcgcagcccggactcggtaatatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccagcggccgcpcr扩增的片段2的序列(seqidno:17)：gcggccgccccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcgagatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacatatgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagccgcggccgcpcr片段3的序列(seqidno:18)：gcggccgctgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgcacggttacgatgcgcccatctacaccaacgtgacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtgatggtgctgcgctggagtgacggcagttatctggaagatcaggatatgtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcgagttgcgtgactacctacgggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgatgagcgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggagcgccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaagcctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatgcggccgcpcr片段4的序列(seqidno:19)：gcggccgcaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcagaacaactttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatgtggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccgatgatccgcgctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaatgaatcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcggcggagccgacaccacggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatggtccatcaaaaaatggctttcgctacctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatccccgtttacagggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggcgatacgccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaacaccagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtgagcggccgcpcr片段5的序列(seqidno:20)：gcggccgcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccgccagtcaggctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgcgatcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattgaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacggcagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtggtcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcaggtagcagagcgggtaaactggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactgatggaaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggattggtggcgacgactcctggagcccgtcagtatcggcggaattccagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaaatgagcggccgc按设计的最终组装的顺序(片段1和2之间、2和3之间、3和4之间、4和5之间)，5个片段各自与相邻片段具有80bp的重叠区。片段1和5还与载体末端共享20bp重叠。可以使用任何可用的载体，例如，pacyc184(newenglandbiolabs,ipswich,ma)。通过反向pcr制备pacyc184载体，其使得可以在用(newenglandbiolabs,ipswich,ma)处理和热灭活之后在上述组装混合物的存在下进行片段1-5的组装(参见图2a-2c)。在组装过程中，从阴影区域延伸的核苷酸通过t5核酸外切酶降解，同时灰色显示的核苷酸通过聚合酶除去。片段组装并转化到大肠杆菌中之后，在具有iptg和x-gal的平板上记录通过蓝色/白色选择确定的富有成效组装。将t5核酸外切酶、taq连接酶、链置换dna聚合酶和ss结合蛋白质(etssb)在缓冲液中合并在反应混合物中，以形成mix1。这些酶均获自newenglandbiolabs,ipswich,ma。将5种150ngnoti-hf-消化的质粒(质粒a、b、c、d和e)与105ng载体以及与mix1或gamm混合成总体积20μl。将反应物在50℃下孵育60分钟。使用2μl组装的产物转化到neb5-α(newenglandbiolabs,ipswich,ma)感受态细胞中。然后将细胞涂布在包含氯霉素的平板上。阳性组装体可以在具有氯霉素+iptg+x-gal的平板上作为蓝色菌落识别出，并在37℃下孵育过夜。转化之前确认所有片段均接合并连接的组装产物的pcr包括以下步骤：在确保5片段和载体连接在一起的pcr中使用1μl组装产物。使用在载体上退火的成对pcr引物扩增整个组装的laclz基因(5.3kb)。条带1和2是复制pcr结果。条带m是来自newenglandbiolabs,ipswich,ma的2-logdna梯状带(参见图4)。测序结果获自选取的8个菌落，并且出于sanger测序的目的将质粒dna提纯。使用6个引物对4.8kb片段进行测序。片段之间的接合序列以及从重叠区的延长区均显示出小于2％的序列错误。用于对组装的dna进行测序的引物：实施例3：将单链寡核苷酸组装到线性化载体或两个不同的dsdna中与用于靶向智人(h.sapiens)的基因的sgrna对应的寡核苷酸设计如下：1.筛选具有所需靶标序列的pam序列。例如，以下中的ngg5’gcgaagaacctcttcccaagangg3’(seqidno:27)2.设计包含两侧为部分u6启动子序列和骨架rna序列的21核苷酸靶标序列的71-碱基ssdna寡核苷酸。参见例如图8a-c，其中ss寡核苷酸确定为：5’atcttgtggaaaggacgaaacaccggcgaagaacctcttcccaagagttttagagctagaaatagcaagtt3’(seqidno:28)或者图9a-c，其中ss寡核苷酸设计成产生以下随机文库：5’atcttgtggaaaggacgaaacaccgn21gttttagagctagaaatagcaagtt3’(seqidno:29)3.在1×nebuffer2(newenglandbiolabs,ipswich,ma)中制备ssdna寡核苷酸，最终浓度为0.2μm。4.形成包含5μlssdna寡核苷酸(0.2μm)、30ng限制性内切酶-线性化载体和ddh2o的10μl反应混合物。5.适合用于上述方法的载体是来自lifetechnology的ds载体(具有ofp报告子的crispr核酸酶载体试剂盒，目录号：a21174)。其他载体如质粒#42230(px330-u6-chimeric_bb-cbh-hspcas9)由addgene提供(具体参见https://www.addgene.org/42230/)。另外可选地，可以使用在u6启动子的控制下包含sgrna骨架的任意质粒。6.将包含ss结合蛋白质、连接酶、核酸外切酶和聚合酶的10μl主体混合物加入到反应混合物中，并将组装反应物在50℃下孵育1小时。7.将neb10-β感受态大肠杆菌遵照制制造商(newenglandbiolabs)的方案用2μl组装产物转化。8.将100μl转化的细胞涂布在具有氨苄青霉素抗生素的平板上，并在37℃下孵育过夜。9.选取10个菌落使其生长，并对质粒dna进行提纯用于测序。与常规的必须合成两种寡核苷酸并将其再退火的克隆方法不同，该实施例提供一种简单的方式来设计寡核苷酸并将其用所需载体进行组装，其相对于常规方法表现出显著的改善，特别是在节约时间、便于使用和成本方面。seqidno:1mildadyitedgkpiirlfkkengrfkveydrnfrpyiyallkddsaiddvrkitserhgkvvrvidvekvkkkflgrpievwklyfehpqdvpamrdkirehpavidifeydipfakrylidkglipmegneeltflavdietlyhegeefgkgpiimisyadeegakvitwkkidlpyvevvaneremikrlikvirekdpdviityngdnfdfpyllkraeklgmklplgrdnsepkmqrlgdslaveikgrihfdlfpvirrtinlptytleavyeaifgkqkekvypheiaeawetgkglervakysmedakvtyelgkeffpmeaqlarlvgqplwdvsrsstgnlvewyllrkayernelapnkpdereyerrlresyeggyvkeperglwegivsldfrslypsiiithnvspdtlnkegcgeydeapevghrfckdfpgfipsllgslleerqkikkrmkeskdpverklldyrqraikilansfygyygyakarwyckecaesvtawgrqyielvrreleergfkvlyidtdglyatipgeknweeikrralefvnyinsklpgileleyegfytrgffvtkkkyalideegkivtrgleivrrdwseiaketqakvleailkhgnveeavkivkevteklsnyeipveklviyeqitrplneykaigphvavakrlaakgikikpgmvigyvvlrgdgpiskraiaieefdgkkhkydaeyyienqvlpaverilkafgykredlrwqktkqvglgawlkvkksseqidno:2iinpqarltpleleileiikqkksititeikeilserrkseyplslvseyisrlerkgyvkkiakgrkkfvealiseqidno:3mildadyitedgkpiirlfkkengrfkveydrnfrpyiyallkddsaiddvrkitserhgkvvrvidvekvkkkflgrpievwklyfehpqdvpamrdkirehpavidifeydipfakrylidkglipmegneeltflavdietlyhegeefgkgpiimisyadeegakvitwkkidlpyvevvaneremikrlikvirekdpdviityngdnfdfpyllkraeklgmklplgrdnsepkmqrlgdslaveikgrihfdlfpvirrtinlptytleavyeaifgkqkekvypheiaeawetgkglervakysmedakvtyelgkeffpmeaqlarlvgqplwdvsrsstgnlvewyllrkayernelapnkpdereyerrlresyeggyvkeperglwegivsldfrslypsiiithnvspdtlnkegcgeydeapevghrfckdfpgfipsllgslleerqkikkrmkeskdpverklldyrqraikilansfygyygyakarwyckecaesvtawgrqyielvrreleergfkvlyidtdglyatipgeknweeikrralefvnyinsklpgileleyegfytrgffvtkkkyalideegkivtrgleivrrdwseiaketqakvleailkhgnveeavkivkevteklsnyeipveklviyeqitrplneykaigphvavakrlaakgikikpgmvigyvvlrgdgpiskraiaieefdgkkhkydaeyyienqvlpaverilkafgykredlrwqktkqvglgawlkvkksgtggggiinpqarltpleleileiikqkksititeikeilserrkseyplslvseyisrlerkgyvkkiakgrkkfvealiseqidno:33mkekapkidalidctyktednraviylyllenilkdrefspyfyvemlkdriekedidkikefllkedllkfvenlevvnktilkkekeivkiiathpqrvpklrkikecdivkeiyehdipfakrylidsdivpmtywdfenrkqvsieipklktvsfdmevynrdtepdpekdpilmasfwddnggkvitykhfdhsnievvnsekdlikkivemlrqydviftyngdnfdfpylkarakiygidiklgrdgeelkikrggmefrsyipgrvhidlypisrrllkltkytledvvynlfgieklkiphtkivdywanndkilieyslqdakythkigkyffplevmfsrivnqtpfeitrmssgqmveyllmknafkenmivpnkpdekeyrkrlltsyeggyvkepekgmfediismdfrchprgtkvivknngltdienvkvgdyvlgidgwqkvkrvwkypyngflvnvnglkstpnhkipvikkengkdrvidvssiyllnlkgckilkiknfesigmfgkifkkdtkikkvkgllekiayidpreglvikvknekedifktvipilkelnilykqvdektiiidsidgllkyivtigfndkneekikeiikeksflefkeledikisieeyegyvydltlegrpyyfangilthnslypsiiiaynispetldcecckdisekilghwfckkreglipktlrglierriniknkmkkmesekeineeynlldyeqrslkilansvygylafprarfysrecaevitylgrkyiletieeaekfgfkviyadsvvkdakviikedgkikeikiedlfkkvdytigdkeycilnnvetltiedtklvwrkvpyimrhrtnkkiyrvkvkdryvditedhsiigvknnklvelkpteikddetkliilnkdlksynfasveeincikysdyvydieventhrffangilvhntdgfyavwkekiskddlikkalefvkyinsklpgtmelefegyfkrgifitkkryalidengrvivkglefvrrdwsnlaritqrrvlealllegdinkakkaiqdvikdlrekkikkedliiytqltknpneykttaphveiakkmmregkkikigdvigyiivkgsksiseraklpeevsieeidvnyyidnqilppvlrimeavgvsknelkkegtqltldrflkseqidno:34mervegwlidadyetiggkavvrlwckddqgifvaydynfdpyfyvigvdedilknaatstrreviklksfekaqlktlgrevegyivyahhpqhvpklrdylsqfgdvreadipfayrylidkdlacmdgiaiegekqggvirsykiekveriprmefpelkmlvfdcemlssfgmpepekdpiivisvktndddeiiltgderkiisdfvkliksydpdiivgynqdafdwpylrkraerwnipldvgrdgsnvvfrggrpkitgrlnvdlydiamrisdikikklenvaeflgtkieiadieakdiyrywsrgekekvlnyarqdaintyliakellpmhyelskmirlpvddvtrmgrgkqvdwlllseakkigeiapnppehaesyegafvleperglhenvacldfasmypsimiafnispdtygcrddcyeapevghkfrkspdgffkrilrmliekrrelkvelknlspesseyklldikqqtlkvltnsfygymgwnlarwychpcaeattawgrhfirtsakiaesmgfkvlygdtdsifvtkagmtkedvdrlidklheelpiqievdeyysaiffvekkryagltedgrlvvkglevrrgdwcelakkvqrevievilkeknpekalslvkdvilrikegkvsleevviykgltkkpskyesmqahvkaalkaremgiiypvsskigyvivkgsgnigdraypidliedfdgenlriktksgieikkldkdyyidnqiipsvlrilerfgyteaslkgssqmsldsffsseqidno:35mikawlldvdyvtendravirlwckddkgvfvaydrnflpyfyvigckaedvmkvkvrtnegiitplkveeieakslgkpikalkvytrhpqhvpklreeikkfaevreadipfayrylidkdlacmdgieiepiavkegvlrayevrsvrrvekkgfpdlkilafdcemlaqfmpdpekdpiiaiavkcgdfeevlhgderdilrrfvsiikeqdpdiivgynqdnfdwpyvkkraekfgirldigrdrseisfrggrpkiagrlnvdlydialkipdvkiktlkkvaeflgakveeediegrdiykcwmrgekekvfkhvlndvlttyrlalellpmhyelsrmirlplddvarlgrgkqvdyfllseakkineiapnppeieesyegafvleparglhenvacldfasmypsiminfnispdtlvkgecedcyvapevghkfrkspdgffkrilkmliekrremkrqmkeldpdsedyklldikqqtlkvltnsfygytgwnlarwycrecaeattawgryfikravkiaesmgfevlygdtdslfikknklnlkdlekeclklidviskelpiqleidefykaiffvekkryagltdddrivvkglevrrgdwcelakrvqrevieiilrernpdkalkfvknvieeikegkfkledyviykgltkkpdkyeskqahvkaalramemgiyypigtkvgfvivkgggsisdraypielieefdgenlkirtpsgimvkkidkdyyidhqiipavmrilerfgyteaslkttiqktlfdftseqidno:36mklvifdgnsilyraffalpelttssniptnaiygfinvilkyleqekpdyiavafdkrgrearkseyqeykanrkpmpdnlqvqipyvreilyalnipivefegyeaddvigslvnkfkntgldiviitgdrdtlqlldknvvvkivstkfdrtmedlytienikekygvwanqvpdykalvgdqsdnipgvkgigeksaqklleeyssleeiyqnldkikgsirekleagkdmaflskrlativcdlplnvnledlrtkewnkerlyeilvqlefksiikrlglseniqfefvqqrtdipdveqrelesisrirskeiplmfvqdekcfylydqesntvfvtrdrhlveeilksdtvkivydlknifhqlnledtdnikncedvmiasyvldstrssyeletlfvsylntdieav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gccgctttacaggcttcg60acgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagat120ttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgcca180atcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagc240tccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttc300accacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgtt360actggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccg420cgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactc480ctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaa540tggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacc600cacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgat660gtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcg720tccggcgtagaggatcgagatctcgatcccgcgaaattaatacgactcactataggggaa780ttgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagata840tacatatgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaa900accctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgta960atagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagccgcggccgc1016<210>18<211>1016<212>dna<213>人工序列<220><223>合成的构建体<400>18gcggccgctgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcacc60gatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttccg120gcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtc180gtcgtcccctcaaactggcagatgcacggttacgatgcgcccatctacaccaacgtgacc240tatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcg300ctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgat360ggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggac420agtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctc480gcggtgatggtgctgcgctggagtgacggcagttatctggaagatcaggatatgtggcgg540atgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgat600ttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagtt660cagatgtgcggcgagttgcgtgactacctacgggtaacagtttctttatggcagggtgaa720acgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgatgagcgtggtggt780tatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggagcgccgaa840atcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaa900gcagaagcctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctg960aacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatgcggccgc1016<210>19<211>1016<212>dna<213>人工序列<220><223>合成的构建体<400>19gcggccgcaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgat60tcgaggcgttaaccgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagac120gatggtgcaggatatcctgctgatgaagcagaacaactttaacgccgtgcgctgttcgca180ttatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatgtggtgga240tgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccgatgatccgcg300ctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcaccc360gagtgtgatcatctggtcgctggggaatgaatcaggccacggcgctaatcacgacgcgct420gtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcggcggagc480cgacaccacggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcc540cttcccggctgtgccgaaatggtccatcaaaaaatggctttcgctacctggagagacgcg600cccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaa660atactggcaggcgtttcgtcagtatccccgtttacagggcggcttcgtctgggactgggt720ggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtga780ttttggcgatacgccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccg840cacgccgcatccagcgctgacggaagcaaaacaccagcagcagtttttccagttccgttt900atccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgataacgagct960cctgcactggatggtggcgctggatggtaagccgctggcaagcggtgagcggccgc1016<210>20<211>1168<212>dna<213>人工序列<220><223>合成的构建体<400>20gcggccgcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgc60tggatggtaagccgctggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaac120agttgattgaactgcctgaactaccgcagccggagagcgccgggcaactctggctcacag180tacgcgtagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggc240agcagtggcgtctggcggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcc300cgcatctgaccaccagcgaaatggatttttgcatcgagctgggtaataagcgttggcaat360ttaaccgccagtcaggctttctttcacagatgtggattggcgataaaaaacaactgctga420cgccgctgcgcgatcagttcacccgtgcaccgctggataacgacattggcgtaagtgaag480cgacccgcattgaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccagg540ccgaagcagcgttgttgcagtgcacggcagatacacttgctgatgcggtgctgattacga600ccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccgga660ttgatggtagtggtcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgc720atccggcgcggattggcctgaactgccagctggcgcaggtagcagagcgggtaaactggc780tcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgct840gggatctgccattgtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgc900gctgcgggacgcgcgaattgaattatggcccacaccagtggcgcggcgacttccagttca960acatcagccgctacagtcaacagcaactgatggaaaccagccatcgccatctgctgcacg1020cggaagaaggcacatggctgaatatcgacggtttccatatggggattggtggcgacgact1080cctggagcccgtcagtatcggcggaattccagctgagcgccggtcgctaccattaccagt1140tggtctggtgtcaaaaatgagcggccgc1168<210>21<211>24<212>dna<213>人工序列<220><223>合成的构建体<400>21aaaaccaccctggcgcccaatacg24<210>22<211>24<212>dna<213>人工序列<220><223>合成的构建体<400>22cccggactcggtaatggcgcgcat24<210>23<211>24<212>dna<213>人工序列<220><223>合成的构建体<400>23ggaagcagcccagtagtaggttga24<210>24<211>24<212>dna<213>人工序列<220><223>合成的构建体<400>24ggtgctgcgctggagtgacggcag24<210>25<211>24<212>dna<213>人工序列<220><223>合成的构建体<400>25cggccaccgatattatttgcccga24<210>26<211>24<212>dna<213>人工序列<220><223>合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