高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代
Summary
使用DNA装配，可以并行地构建在单个克隆反应多个CRISPR矢量，使得大量CRISPR的建设矢量a简单的任务。番茄毛状根是一个很好的模型系统来验证CRISPR载体和产生突变体材料。
Abstract
Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.
Introduction
生成具有针对性的CRISPR DNA修改的能力/ Cas9有功能基因组学研究的巨大潜力。还有CRISPR / Cas9系统的两个组件;的Cas9核酸酶，来自金化脓和指示Cas9到目标DNA的网站，1的约100-nt的导的RNA（gRNA）分子衍生。目标识别由第一赋予〜了gRNA，其允许高通量生产靶向载体2,3的20-nt的。可工程改造大多数生物体，已经已经与CRISPR / Cas9技术4,5。
在植物中，组成型启动子，如CaMV 35S启动子，通常用于驱动Cas9核酸6的表达。的gRNAs使用RNA聚合酶III U6或U3启动子限制gRNA的第一基表示到一个G，对于U6，或A为U3，对有效转录。然而RNA聚合酶II舞会oters，这是自由的这些限制，也已经使用7,8-。
不同gRNAs诱导不同的效率DNA突变，并且因此它可以在整个植物转化的投资或设立广泛的表型屏幕之前先验证CRISPR载体是重要的。 CRISPR的瞬时表达构建体在植物中，通过使用例如农杆菌渗入，通常导致DNA修饰的一个较低的频率相比，稳定的植物6，使得突变困难和表型分析不切实际用这样的方法进行检测。所谓毛状根是由于能够在几周内产生大量的独立的，稳定转化的材料，而不是个月稳定的植物一个方便的替代系统。 CRISPR载体在发根诱导9,10 DNA突变非常有效。
DNA组装方法有效地结扎含，可欣赏的DNA片段apping结束11。的一些DNA的装配方法的主要优点是将单链DNA（ 即寡核苷酸）插入组装产品的能力。因为gRNAs仅〜20-nt的长和新的目标可以用合成的寡核苷酸进行，这些DNA装配方法非常适合于CRISPR克隆。这里所述的协议是基于P201系列CRISPR载体已在大豆10，杨树12被成功地使用，现在番茄。提出的克隆程序在当前克隆方法10提供了几个优点。即，可以在一个单一的一天的单个克隆反应中产生完全功能的载体。载体的构建也可以合并，以产生多个并行CRISPR载体，进一步减少操作时间和材料成本。我们还提出了一个协议番茄毛根的产生作为一种有效的方法，以产生具有目标基因缺失的转基因材料。发根是用来有效吃了CRISPR载体和为后续实验提供材料。
Protocol
Representative Results
Discussion
Since DNA assembly is used to recombine any overlapping DNA sequences, this cloning method can be applied to any CRISPR vector construction. Most CRISPR cloning schemes use either gene synthesis of the gRNA, type IIS restriction enzymes17,18, or overlap-extension PCR19. Each of these techniques has inherent advantages and disadvantages, but they typically require multiple hands-on cloning steps. The primary advantage of the cloning method presented here is that the entire process occurs in a single,…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是由美国国家科学基金会资助IOS-1025642（GBM）的支持。我们感谢玛丽亚·哈里森提供ARqua1应变。
Materials
| NEBuilder® (HiFi DNA assembly mix) | New England Biolabs | E5520 | |
| p201N:Cas9 | Addgene | 59175 | The p201H:Cas9 plasmid (59176) is also compatible with the reported overlaps and enzymes. |
| pUC gRNA Shuttle | Addgene | 47024 | |
| SwaI | New England Biolabs | R0604S | |
| SpeI | New England Biolabs | R0133S | |
| Zymo clean and concentrator-5 column purification | Zymo Research | D4003 | |
| NEB Buffer 2.1 | New England Biolabs | B7202S | |
| NEB CutSmart (Buffer 4) | New England Biolabs | B7204S | |
| NEB Buffer 3.1 | New England Biolabs | B7203S | |
| EconoSpin Mini Spin Column (plasmid prep) | Epoch Life Sciences | 1910-050/250 | |
| EcoRV-HF® | New England Biolabs | R3195S | |
| StyI-HF® | New England Biolabs | R3500S | |
| MS Salts + Gamborg Vitamins | Phytotechnology Laboratories | M404 | |
| Phytagel™ (gellan gum) | Sigma Aldrich | P8169 | |
| GA-7 Boxes | Sigma Aldrich | V8505 | |
| Micropore™ surgical tape | 3M | 1535-0 | |
| Timentin® (Ticarcillin/Clavulanic acid) | Various | 0029-6571-26 | |
| Primers 5' -> 3' | |||
| SwaI_MtU6F | GATATTAATCTCTTCGATGAAATTTATGCCTATCTTATATGATCAATGAGG | ||
| MtU6R | AAGCCTACTGGTTCGCTTGAAG | ||
| ScaffoldF | GTTTTAGAGCTAGAAATAGCAAGTT | ||
| SpeI_Scaffold R | GTCATGAATTGTAATACGACTCAAAAAAAAGCACCGACTCGGTG | ||
| StUbi3P218R | ACATGCACCTAATTTCACTAGATGT | ||
| ISceIR | GTGATCGATTACCCTGTTATCCCTAG | Cannot be used for Sanger sequencing since there is a second binding site on the plasmid | |
| UNS1_Scaffold R | GAGAATGGATGCGAGTAATGAAAAAAAGCACCGACTCGGTG | ||
| UNS1_MtU6 F | CATTACTCGCATCCATTCTCATGCCTATCTTATATGATCAATGAGG | ||
| p201R | CGCGCCGAATTCTAGTGATCG | ||
| Bolded sequences denote 20-nt overlaps with linearized p201N:Cas9. | |||
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