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

Summary

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.

Abstract

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' 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. 

Introduction

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 humans^1,2, 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 genes^4,5.

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 on^7,8. 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′ 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′ and 3′ 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′ and 3′ 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.

Protocol

1. Determination of optimal Cas9/sgRNA cleavage site upstream of cDNA/ORF

1. Preparation of cDNA or ORF plasmids with identical vector backbone
   1. Purchase cDNA/ORF clones that are available in public resources, such as the Drosophila genome resource center (DGRC, https://dgrc.bio.indiana.edu/) Gold Collection^10,11, the mouse mammalian gene collection (MGC, https://genecollections.nci.nih.gov/MGC/), and the human CCSB-broad Lentiviral expression library¹². In this protocol, we take as an example the pOT2 vector-based cDNA/ORF clones, which are available in the DGRC Gold Collection.
   2. Extract plasmid DNA using a plasmid mini-prep kit. Measure the concentration of plasmids with a spectrophotometer.
2. Design of sgRNAs
   1. Visit the CHOPCHOP website (https://chopchop.cbu.uib.no/). Click the button Paste Target and then input the DNA sequence of interest. The sequence of interest is a 20-100 bp region located in the pOT2 vector backbone upstream of the cDNA/ORF 5' end.
   2. Choose the species Drosophila melanogaster. Click the button Find Target Sites. Choose sgRNA candidates with a predicted efficiency higher than 80%.
3. Preparation of sgRNAs
   1. Order PAGE-purified oligos, a forward primer (5′ -TAATACGACTCACTATAGG(N)₂₀GTTTTAGAGCTA
      GAAATAG-3′) where (N)₂₀ is the target sequence of a sgRNA, and a sgRNA scaffold reverse primer sgRNA-REV (5′-AAAAGCACCGACTCGGTGCCACTT-3′).
      NOTE: Recommend using PAGE-purified high-quality oligonucleotides.
   2. Prepare a DNA template by PCR for in vitro transcription (IVT) of sgRNA. Set up a 100 µL PCR reaction as follows: 5 µL of template pX330 (Addgene plasmid 42230, a gift from Feng Zhang; 0.1 ng/µL), 5 µL of forward primer (10 µM), 5 µL of sgRNA-REV (10 µM), 50 µL of 2x High fidelity PCR master mix, and 35 µL of diethyl pyrocarbonate (DEPC)-treated water in a PCR tube.
   3. Perform PCR using the following thermocycling conditions: 1 cycle of 98 °C for 30 s; 5 cycles of 98 °C for 10 s, 51 °C for 10 s, 72 °C for 5 s; 30 cycles of 98 °C for 10 s, 72 °C for 15 s; 1 cycle of 72 °C for 2 min. Incubate reactions in a thermal cycler.
   4. Separate PCR products by 0.8% agarose gel electrophoresis. A ~120-bp DNA fragment should be visible on the gel under UV. Purify the ~120-bp DNA fragment using a gel extraction kit. The purified DNA fragment serves as a template for IVT of sgRNA.
   5. Set up a 5 µL IVT reaction as follows: 165 ng of ~120-bp purified gel DNA fragment, 1.65 µL of NTP buffer mix, 0.33 µL of T7 RNA polymerase mix, and DEPC-treated water. Incubate reactions at 37 °C for 4 h. Follow the manual instructions of an in vitro transcription kit.
   6. Add 45 µL of DEPC-treated water to the IVT reaction product. Treat the IVT reaction products as RNA and use DEPC-treated water as a blank control. Measure the concentration of sgRNA with a spectrophotometer. The concentration of diluted sgRNA is around 870 ng/µL.
   7. Dilute the IVT reaction product to 20 ng/µL with DEPC-treated water. Aliquot and store sgRNA directly at -80 °C.
      NOTE: After the IVT reaction, removal of the DNA template by DNase digestion is not necessary for measuring the precise concentration of sgRNA, as the amount of the DNA template is less than 0.4% of that of sgRNA and the DNA template does not affect subsequent CRISPRmass reaction. Therefore, sgRNA purification is not necessary.
4. Evaluation of sgRNAs
   1. Digest a cDNA/ORF plasmid bearing pOT2 vector backbone with a restriction enzyme. Separate the resulting DNA fragments by 0.8% agarose gel electrophoresis. Purify the DNA substrate with a gel extraction kit.
      NOTE: A resulting DNA fragment containing the sgRNA targeting sequences of the pOT2 vector backbone serves as the DNA substrate of Cas9/sgRNAs. The cleavage sites of Cas9/sgRNAs are asymmetrically located in the DNA substrate, and thereby, cleavage of the DNA substrate by Cas9/sgRNAs yields two DNA fragments of different sizes, which makes them easier to distinguish from the uncleaved DNA substrate in the digestion reaction.
   2. Set up a 5 µL Cas9 cleavage reaction as follows: 0.2 µL of 1.22 µM S. pyogenes Cas9, 0.5 µL of 20 ng/µL sgRNA, 0.015 pmol of DNA substrate, 0.5 µL of 10x Cas9 Buffer, and DEPC-treated water. Incubate reactions at 37 °C for 1 h.
   3. Separate the cleavage products by 0.8% agarose gel electrophoresis. Load the intact DNA substrate as a negative control for the digested DNA substrate.
      NOTE: Here, intact DNA substrate refers to DNA substrate that is not subjected to digestion reactions by Cas9/sgRNAs. Cleavage of the DNA substrate by Cas9/sgRNAs yields two DNA fragments of different sizes, which makes them easier to distinguish from the uncleaved DNA substrate in the digestion reaction.
   4. Analyze cleavage patterns by a digital imaging system (Figure 2). Select the most efficient sgRNA for subsequent CRISPRmass experiments. Figure 2 shows that all four sgRNAs exhibit high cleavage efficiency and can be used for CRISPRmass. Select the underlined sgRNA for subsequent experiments.
      NOTE: The less the uncleaved DNA substrate is, the more efficient the sgRNA is for Cas9/sgRNA digestion of DNA substrate.

2. Construction of a UAS module

NOTE: A UAS module comprises a core UAS and around 20-40 bp of the 5′ and 3′ terminal sequences overlapping with 5′ and 3′ ends of the backbone cleavage site, respectively (Figure 1). The 5′ and 3′ terminal sequences of the UAS module are determined by the backbone cleavage site of the pOT2 vector.

1. Clone the pOT2 vector-specific UAS module into the EcoRI site of the pCR8GW vector, resulting in pCR8GW-Amp-W-attB-UAS-Hsp70 plasmid (Supplementary File 1). Release the pOT2 vector-specific UAS module by digestion of pCR8GW-Amp-W-attB-UAS-Hsp70 plasmid with EcoRI at 37 °C for 1 h.
2. Separate the cleavage products by 0.7% agarose gel electrophoresis. Purify the 6178-bp UAS module with a gel extraction kit. Dilute the UAS module to 23 ng/µL for subsequent experiments. This 6178-bp fragment serves as a UAS module for the construction of UAS-cDNA/ORF plasmids from pOT2 vector-based cDNA/ORF clones.

3. Large-scale construction of UAS-cDNA/ORF plasmids by CRISPRmass

NOTE: CRISPRmass is standardized as massively parallel two-step test tube reactions prior to E. coli transformation (Figure 1).

1. Test tube reaction step 1
   1. Cleave the identical vector backbones of the cDNA/ORF plasmids by Cas9/sgRNA. Arrange 0.2 mL tubes in an aluminum cooling block on ice and label the tubes carefully. Prepare the master mix as follows.
      1. For a single reaction, add 0.16 µL of 1.22 µM S. pyogenes Cas9, 0.4 µL of 20 ng/µL sgRNA, 0.24 µL of 10x Cas9 buffer, 1.2 µL of DEPC-treated water. For N reactions, add (N+1) x 0.16 µL of 1.22 µM S. pyogenes Cas9, (N+1) x 0.4 µL of 20 ng/µL sgRNA, (N+1) x 0.24 µL of 10x Cas9 buffer, (N+1) x 1.2 µL of DEPC-treated water.
   2. Vortex, mix well, and spin down. Aliquot 2 µL of master mix to each tube. Add 0.4 µL of 0.019 µM of cDNA/ORF plasmid to each tube. Incubate cleavage reactions at 37 °C for 1 h.
      NOTE: Dilute each plasmid to 0.019 µM with DEPC-treated water. If the concentration of a plasmid is lower than 0.019 µM, add plasmid DNA directly to the cleavage reaction. Use RNase-free tubes and DEPC-treated water. Performing these steps on a large scale will take several hours. Unless otherwise specified, keep reagents and reactions on ice.
2. Test tube reaction step 2
   1. Insert a vector-specific UAS module from step 2 into the Cas9-linearized cDNA/ORF plasmids by single step reaction assembly. Set up a 1.4 µL single step reaction for each sample. Arrange the 0.2 mL tubes in an aluminum cooling block on ice and label the tubes carefully.
      1. Prepare the master mix as follows. For a single reaction, add 0.07 µL of 0.006 µM UAS module and 0.7 µL of single step reaction assembly master mix. For N reactions, add (N+1) x 0.07 µL of 0.006 µM UAS module and (N+1) x 0.7 µL of single step reaction assembly master mix.
   2. Vortex, mix well, and spin down. Aliquot 0.77 µL of the master mix to each tube. Add 0.7 µL of Cas9-linearized cDNA/ORF plasmid to each tube. Incubate reactions at 50 °C for 1 h.
      NOTE: Purification of single step assembly reaction products is not necessary. Performing these steps on a large scale takes several hours. Unless otherwise specified, keep reagents and reactions on ice.
3. Transform single step assembly reaction products into E. coli on a large scale.
   1. Prechill 10 µL tips at 4 °C. Prechill 1.5 mL tubes in an aluminum cooling block on ice.
   2. Thaw competent E. coli cells on ice for 5 min. Aliquot 10 µL of competent cells to each prechilled 1.5 mL tube.
      NOTE: Use commercially available competent E. coli cells with a transformation efficiency of at least 1 x 10⁸ CFU/µg of pUC19 DNA.
   3. Add 1 µL of single step assembly reaction product to 10 µL of competent cells, swirl gently to mix, and place on ice for 30 min.
   4. Incubate the tubes in a 42 °C water bath for 1 min, and then immediately chill on ice for 2 min.
   5. Add 100 µL of SOC medium (prewarmed to 37 °C) into each tube at room temperature, and incubate in a shaking incubator (250 rpm) at 37 °C for 1 h.
   6. Warm up to 37 °C the LB plates that contain an antibiotic corresponding to the antibiotic resistance gene of the UAS module. Spread cells onto the plates and incubate at 37 °C overnight.
      NOTE: Performing these steps on a large scale takes several hours. Unless otherwise specified, keep reagents and reactions on ice.
4. Verify UAS-cDNA/ORF plasmids constructed by CRISPRmass
   1. Pick a single colony and inoculate it in 4.5 mL of LB liquid medium supplemented with the appropriate selection of antibiotics corresponding to the antibiotic resistance gene of the UAS module. Incubate overnight at 37 °C while shaking at 250 rpm.
   2. Extract plasmid DNA by using a plasmid mini-prep kit. Set up a 20 µL restriction digestion reaction as follows: 300-500 ng/µL of plasmid, appropriate restriction enzyme(s), restriction digestion buffer, and ultrapure water. Incubate reactions at 37 °C for 1 h.
   3. Separate DNA fragments by 0.8% agarose gel electrophoresis and analyze by a digital imaging system (Figure 3).

Results

We applied CRISPRmass to generate a genome-wide UAS-cDNA/ORF plasmid library using 3402 cDNA/ORF clones bearing pOT2 vector backbone from the DGRC Gold Collection. We randomly analyzed only one colony for each UAS-cDNA/ORF construct, and subsequent restriction analysis with PstI indicated that 98.6% of UAS-cDNA/ORF constructs were created successfully⁹. The rationale for using PstI for restriction analysis of UAS-cDNA/ORF constructs bearing pOT2 vector backbone is as follows. There are two PstI sites in the UAS module for the pOT2 vector-based cDNA/ORF clones, and there is a PstI site in the pOT2 vector backbone. Thereby, digestion of a pOT2 vector-based UAS-cDNA/ORF construct with PstI yields a 408 bp UAS module-specific fragment and a 5649 bp UAS module-vector assembly-specific fragment. The 408 bp and 5649 bp PstI fragments indicate that UAS-cDNA/ORF constructs for pOT2 vector-based cDNA/ORF clones are created successfully, and the representative results are shown in Figure 3.

Figure 1: Flowchart of UAS-cDNA/ORF library construction using CRISPRmass. The CRISPRmass pipeline for construction of a UAS-cDNA/ORF library. (1) The first step test tube reaction. The identical vector backbones of cDNA/ORF plasmids are cleaved by Cas9/sgRNA. The cleavage site is in vector backbones adjacent upstream of the cDNA/ORF 5′ end. The cleavage products, without purification, are directly subjected to the second step of the test tube reaction. The sgRNAs used in cleavage reactions are prepared by in vitro transcription and can be used directly without purification. Then, 28-40 bp of the 5′ end of the cleavage site (yellow box) is defined as 5′ end overlap and 28-40 bp of the 3′ end of the cleavage site (red box) is defined as 3′ end overlap. The vector backbone carries the antibiotic A resistance gene (green box). (2) The second step is the test tube reaction. A vector specific UAS module is joined into the Cas9-linearized cDNA/ORF plasmids right upstream of the cDNA/ORF5′ end through single step reaction assembly, resulting in UAS-cDNA/ORF constructs. Single step reaction assembly products are directly subjected to E. coli transformation. Transformants are selected on LB agar plates containing antibiotic B corresponding to the antibiotic B resistance gene (brown box) of the vector specific UAS module. Only the desired UAS-cDNA/ORF colonies can grow. A UAS module comprises 10 copies of UAS, an Hsp70 minimal promoter, an attB sequence for phiC31-mediated genomic integration, a mini-white transformation marker for Drosophila transgenesis, a selectable antibiotic B resistance gene for positive selection, and the 5′ end overlap and 3′ end overlap enabling single step reaction assembly. Single step reaction assembly filters out any potential off-target DNA cleavages caused by CRISPR/Cas9. This figure has been modified from⁹. Please click here to view a larger version of this figure.

Figure 2: Evaluation of sgRNAs targeting specific regions of pOT2 vector backbone by in vitro Cas9 cleavage analysis. The underlined sgRNAs are selected for CRISPRmass. M is the DNA marker. This figure has been modified from⁹. Please click here to view a larger version of this figure.

Figure 3: Restriction analysis of 20 UAS-cDNA/ORF constructs generated by CRISPRmass. The constructs were analyzed by PstI digestion. The expected restriction patterns for all the UAS-cDNA/ORF constructs were observed. M is the DNA marker. This figure has been modified from⁹. Please click here to view a larger version of this figure.

Supplementary File 1: Construction of pCR8GW-Amp-W-attB-UAS-Hsp70 plasmid. The pOT2 vector-specific UAS module containing plasmid pCR8GW-Amp-W-attB-UAS-Hsp70 is constructed based on three plasmids, pMartini-Amp, pBS-attB-UAS-Hsp70, and pCR8GW-Amp-attB-UAS-Hsp70. Please click here to download this File.

Discussion

The most critical steps of CRISPRmass are the design of sgRNAs and the evaluation of sgRNAs. Selection of highly efficient sgRNAs for Cas9 is key to the success of CRISPRmass. If very few or no colonies are observed on the majority of antibiotic-containing LB plates after the transformation of E. coli with single-step reaction assembly products, check plasmid digestion by agarose gel electrophoresis. If plasmids are not well digested, check Cas9 activity, sgRNA degradation and plasmid quality; if plasmids are well digested, check single step reaction assembly reagents and make sure the transformation efficiency of competent cells is at least 1 x 10⁸ cfu/µg of pUC19 DNA. Noteworthily, using an aluminum cooling block can ease large-scale bacterial transformation.

The limitations of CRISPRmass arise from its manipulation and incorporation of vector backbone sequences, which limits the possibility of tagging the cDNAs and ORFs at either 5' or 3' end.

Unlike all the other existing methods^7,8, CRISPRmass manipulates and incorporates vector backbone sequences⁹. Thus, once UAS modules are designed and prepared, CRISPRmass needs no individualized design or manipulation for every single plasmid but massively parallel two step test-tube reactions prior to bacterial transformation, obviating PCR, and its related manipulations. Furthermore, both the in vitro transcribed sgRNAs and single step reaction assembly products do not need to be purified for the subsequent experiments. Compared to Gateway cloning technology, CRISPRmass is more suitable for generating UAS-cDNA/ORF constructs, particularly from long or GC-rich cDNAs/ORFs, as CRISPRmass does not PCR amplify the cDNAs or ORFs themselves⁹.

CRISPRmass can be used for the high-throughput construction of a UAS-cDNA/ORF plasmid library as well as for editing various genome-wide plasmid libraries. CRISPRmass can be applied to the construction of an expression plasmid library by insertion of a CMV promoter into the shared vector sequences adjacent to the 5' end of cDNAs or ORFs. CRISPRmass promises to accelerate the development of functional genomics.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aluminum Cooling Block	Aikbbio	ADMK-0296	To perform bacterial transformation
DEPC-Treated Water	Invitrogen	AM9906
Gel Extraction Kit	Omega	D2500	To purify DNA from agarose gel
Gel Imaging System	Tanon	2500B
HiScribe T7 Quick High Yield RNA Synthesis Kit	NEB	E2050
NEBuilder HiFi DNA Assembly Master Mix	NEB	E2621
Plasmid Mini Kit	Omega	D6943	To isolate plasmid DNA from bacterial cells
Q5 Hot Start High-Fidelity 2x Master Mix	NEB	M0494
S. pyogenes Cas9	GenScript	Z03386
Shaking Incubator	Shanghai Zhichu	ZQLY-180V
T series Multi-Block Thermal Cycler	LongGene	T20	To perform PCR
Trans10 Chemically Competent Cell	TranGen BioTech	CD101
Ultraviolet spectrophotometer	Shimadzu	BioSpec-nano	To measure concentration of DNA or RNA