large-scale construction of uas-cdna/orf plasmids by crispr-based modular assembly (crisprmass)

Построение высокопроизводительной библиотеки плазмид UAS-кДНК/ORF с использованием CRISPRmass

Unbiased whole-genome genetic screening is a powerful approach for identifying genes involved in a given biological process and elucidating its mechanism. Therefore, it is widely used in various fields of biological research. Approximately 60% of Drosophila genes are conserved in humans1,2, and ~75% of human disease genes have homologs in Drosophila3. Genetic screening is mainly divided into two types: loss of function (LOF) and gain of function (GOF). LOF genetic screens in Drosophila have played a critical role in elucidating mechanisms that govern nearly every aspect of biology. However, the majority of Drosophila genes do not have obvious LOF phenotypes4, and therefore, GOF screening is an important method for studying the function of those genes4,5.
The binary GAL4/UAS system is commonly used for tissue-specific gene expression in Drosophila6. In this system, the tissue specifically expresses yeast transcription activator GAL4 that binds to the GAL4 responsive element (UAS) and thereby activates transcription of the downstream genetic components (e.g., cDNA and ORF)6. To perform genome-wide GOF screens in Drosophila, we need to construct a genome-wide UAS-cDNA/ORF plasmid library and, subsequently, a transgenic UAS-cDNA/ORF library in Drosophila.
Construction of a genome-wide UAS-cDNA/ORF plasmid library by conventional methods from publicly available cDNA/ORF clones is time-consuming and laborious, as every gene requires individualized designs, including primer design and synthesis, polymerase chain reaction (PCR), and gel purification, sequencing, restriction digestion, and so on7,8. Therefore, the construction of such a plasmid library is a rate-limiting step in creating a genome-wide transgenic UAS-cDNA/ORF library in Drosophila. Recently, we successfully solved this problem by developing a novel method, CRISPR-based modular assembly (CRISPRmass)9. The core of CRISPRmass is to manipulate the shared vector sequences of a plasmid library through a combination of gene editing technology and seamless cloning technology.
Here, we present a protocol for CRISPRmass, which includes massively parallel two-step test tube reactions followed by bacterial transformation. CRISPRmass is a simple, fast, efficient, and cost-effective method that, in principle, can be used for high-throughput construction of various plasmid libraries.
CRISPRmass strategy 
The procedure of CRISPRmass starts with parallel two-step test tube reactions prior to Escherichia coli (E. coli) transformation (Figure 1). Step 1 is the cleavage of the identical vector backbones of the cDNA/ORF plasmids by Cas9/sgRNA. An ideal cleavage site is adjacent to the 5&#8242; end of cDNA/ORF. The cleavage products do not have to be purified. Step 2 is the insertion of a vector-specific UAS module into the Cas9/sgRNA linearized cDNA/ORF plasmids by Gibson assembly (hereafter referred to as single step reaction), resulting in UAS-cDNA/ORF plasmids. The 5&#8242; and 3&#8242; terminal sequences of a UAS module overlap with those of the linearized cDNA/ORF plasmids, enabling the single step reaction.
The single step reaction products are directly subjected to E. coli transformation. Theoretically, only the desired UAS-cDNA/ORF colonies can grow on Luria-Bertani (LB) plates that contain selection antibiotics corresponding to the antibiotic resistance gene of the UAS module. The UAS module is composed of a core UAS module, an antibiotic resistance gene distinct from that of cDNA or ORF plasmids, and the 5&#8242; and 3&#8242; terminal sequences. A core UAS module comprises 10 copies of UAS, an Hsp70 minimal promoter, an attB sequence for phiC31-mediated genomic integration, and a mini-white transformation marker for Drosophila7.

В центре внимания автора: Упрощение построения полногеномной библиотеки плазмид с помощью CRISPRmass

Модульная сборка на основе CRISPR для высокопроизводительного построения библиотеки плазмид UAS-cDNA/ORF

Functional genomic screening offers a powerful approach to probe gene function, and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. To address this question, we develop a simple and efficient method, CRISPR-based modular assembly, or CRISPRmass.Through cleavage of shared vector sequences of cDNA or ORF plasmid by CRISPR/Cas9 and subsequent insertion of UAS modules by adjacent assembly, the procedure of construction of a genome-wide plasmid library by CRISPRmass is standardized as massively parallel two-step test tube reactions before bacterial transformation. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. It can also be applied to editing various genome-wide plasmid libraries in general, and it's an alternative to gateway technology in high-throughput plasmid library editing, paving the way for the global studies of biological networks.

Крупномасштабное конструирование плазмид UAS-кДНК/ORF с помощью модульной сборки на основе CRISPR (CRISPRmass)

Чтобы провести параллельные двухступенчатые реакции в пробирке модульной сборки на основе CRISPR или CRISPRmass, приступайте к расщеплению идентичных векторных остов плазмид кДНК/ORF с помощью sgRNA Cas9 на первом этапе реакции в пробирке. Для этого расположите правильно маркированные 0,2 миллилитровые трубки в алюминиевом охлаждающем блоке на льду. Приготовьте мастер-смесь для N числа реакций, смешав соответствующие количества Cas9, sgRNA, буфера Cas9 и воды, обработанной DEPC.После того, как компоненты мастер-микса сложены вместе, сделайте их вихревыми, чтобы обеспечить правильное смешивание. Затем раскрутите смесь и добавьте по два микролитра мастер-смеси в каждую тюбик объемом 0,2 миллилитра. Затем добавьте в каждую пробирку 0,4 микролитра 0,019 микромолярной кДНК/ORF плазмиды.Инкубируйте реакции расщепления при 37 градусах Цельсия в течение одного часа. В следующей реакции в пробирке, на втором этапе, приступают к вставке векторно-специфичного модуля UAS в полученные линеаризованные кДНК/ORF плазмиды Cas9. Чтобы настроить одноступенчатую реакцию объемом 1,4 микролитра для каждого образца, расположите маркированные пробирки объемом 0,2 миллилитра в алюминиевом охлаждающем блоке на льду.Подготовьте мастер-микс для N реакций, смешав соответствующие количества модуля UAS и мастер-микс для одноступенчатой реакционной сборки. Хорошо перемешайте смесь перед тем, как вращать ее. Затем нанесите по 0,77 микролитра мастер-смеси на каждую трубку.Добавьте 0,7 микролитра линеаризованной кДНК/ORF плазмиды Cas9 в аликвоту в каждой пробирке и инкубируйте реакции при 50 градусах Цельсия в течение одного часа.

large-scale-construction-uas-cdnaorf-plasmids-crispr-based-modular

Research

JoVE Journal

Genetics

Large-Scale Construction of UAS-cDNA/ORF Plasmids by CRISPR-Based Modular Assembly (CRISPRmass)

CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. Therefore, we recently developed a simple and efficient method, CRISPR-based modular assembly (CRISPRmass), for high-throughput construction of a genome-wide upstream activating sequence-complementary DNA/open reading frame (UAS-cDNA/ORF) plasmid library. Here, we present a protocol for CRISPRmass, taking as an example the construction of a GAL4/UAS-based UAS-cDNA/ORF plasmid library. The protocol includes massively parallel two-step test tube reactions followed by bacterial transformation. The first step is to linearize the existing complementary DNA (cDNA) or open reading frame (ORF) cDNA or ORF library plasmids by cutting the shared upstream vector sequences adjacent to the 5&#39; end of cDNAs or ORFs using CRISPR/Cas9 together with single guide RNA (sgRNA), and the second step is to insert a UAS module into the linearized cDNA or ORF plasmids using a single step reaction. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. The UAS-cDNA/ORF plasmid library can be utilized for gain-of-function screening in cultured cells and for constructing a genome-wide transgenic UAS-cDNA/ORF library in Drosophila.&#160;

We present a protocol for CRISPR-based modular assembly (CRISPRmass), a method for high-throughput construction of UAS-cDNA/ORF plasmid library in Drosophila using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various plasmid libraries.

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. ...

Large-Scale Transformation of UAS-cDNA/ORF Plasmids from CRISPR-Based Modular Assembly into E. coli

To transform the UAS cDNA/ORF plasmids products obtained from the CRISPR based modular assembly into Escherichia coli, pre-chill 10 microliter tips at four degrees Celsius. Also, pre-chill 1.5 milliliter tubes in an aluminum cooling block on ice. After thawing competent E.coli cells on ice for five minutes, add an aliquot of 10 microliters of the cells to each pre-chilled 1.5-milliliter tube.Then add one microliter of the UAS cDNA/ORF plasmid products to each tube containing 10 microliters of competent cells. Swirl the tube gently to mix and place it on ice for 30 minutes. Incubate the tubes at 42 degrees Celsius in a water bath for one minute.Then immediately chill on ice for two minutes. Add 100 microliters of SOC medium, prewarmed to 37 degrees Celsius, to each tube at room temperature and incubate the tubes in a shaking incubator at 250 RPM and 37 degrees Celsius for one hour. Next, warm the LB plates containing an antibiotic corresponding to the UAS module's antibiotic resistance gene to 37 degrees Celsius.Spread the cells onto the warm plates and incubate them at 37 degrees Celsius overnight.