Name: CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library (Video) | JoVE Journal
Uploaded: 2024-05-17T14:35:00.000+00:00
Duration: 5 min 30 s
Description: Learn CRISPR-based modular assembly for high-throughput construction of UAS-cDNA/ORF plasmid libraries in Drosophila. This streamlined method enables efficient genome-wide functional genomic screening.

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5 minutes

This work was sponsored by the National Natural Science Foundation of China (32071135 and 31471010), the Shanghai Pujiang Program (14PJ1405900), and the Natural Science Foundation of Shanghai (19ZR1446400).

1. Determination of optimal Cas9/sgRNA cleavage site upstream of cDNA/ORF
<ol>
	<li>Preparation of cDNA or ORF plasmids with identical vector backbone
	<ol>
		<li>Purchase cDNA/ORF clones that are available in public resources, such as the Drosophila genome resource center (DGRC, https://dgrc.bio.indiana.edu/) Gold Collection10,11, 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.</li>
		<li>Extract plasmid DNA using a plasmid mini-prep kit. Measure the concentration of plasmids with a spectrophotometer.</li>
	</ol>
	</li>
	<li>Design of sgRNAs
	<ol>
		<li>Visit the CHOPCHOP website (https://chopchop.cbu.uib.no/). Click the button&#160;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&#39; end.</li>
		<li>Choose the species Drosophila melanogaster. Click the button Find Target Sites. Choose sgRNA candidates with a predicted efficiency higher than 80%.</li>
	</ol>
	</li>
	<li>Preparation of sgRNAs
	<ol>
		<li>Order PAGE-purified oligos, a forward primer (5&#8242; -TAATACGACTCACTATAGG(N)20GTTTTAGAGCTA 
		GAAATAG-3&#8242;) where (N)20 is the target sequence of a sgRNA, and a sgRNA scaffold reverse primer sgRNA-REV (5&#8242;-AAAAGCACCGACTCGGTGCCACTT-3&#8242;). 
		NOTE: Recommend using PAGE-purified high-quality oligonucleotides.</li>
		<li>Prepare a DNA template by PCR for in vitro transcription (IVT) of sgRNA. Set up a 100 &#181;L PCR reaction as follows: 5 &#181;L of template pX330 (Addgene plasmid 42230, a gift from Feng Zhang; 0.1 ng/&#181;L), 5 &#181;L of forward primer (10 &#181;M), 5 &#181;L of sgRNA-REV (10 &#181;M), 50 &#181;L of 2x High fidelity PCR master mix, and 35 &#181;L of diethyl pyrocarbonate (DEPC)-treated water in a PCR tube.</li>
		<li>Perform PCR using the following thermocycling conditions: 1 cycle of 98 &#176;C for 30 s; 5 cycles of 98 &#176;C for 10 s, 51 &#176;C for 10 s, 72 &#176;C for 5 s; 30 cycles of 98 &#176;C for 10 s, 72 &#176;C for 15 s; 1 cycle of 72 &#176;C for 2 min. Incubate reactions in a thermal cycler.</li>
		<li>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.</li>
		<li>Set up a 5 &#181;L IVT reaction as follows: 165 ng of ~120-bp purified gel DNA fragment, 1.65 &#181;L of NTP buffer mix, 0.33 &#181;L of T7 RNA polymerase mix, and DEPC-treated water. Incubate reactions at 37 &#176;C for 4 h. Follow the manual instructions of an in vitro transcription kit.</li>
		<li>Add 45 &#181;L 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/&#181;L.</li>
		<li>Dilute the IVT reaction product to 20 ng/&#181;L with DEPC-treated water. Aliquot and store sgRNA directly at -80 &#176;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&#160;the DNA template does not affect subsequent CRISPRmass reaction. Therefore, sgRNA purification is not necessary.</li>
	</ol>
	</li>
	<li>Evaluation of sgRNAs
	<ol>
		<li>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.</li>
		<li>Set up a 5 &#181;L Cas9 cleavage reaction as follows: 0.2 &#181;L of 1.22 &#181;M S. pyogenes Cas9, 0.5 &#181;L of 20 ng/&#181;L sgRNA, 0.015 pmol of DNA substrate, 0.5 &#181;L of 10x Cas9 Buffer, and DEPC-treated water. Incubate reactions at 37 &#176;C for 1 h.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
2. Construction of a UAS module
NOTE: A UAS module comprises a core UAS and around 20-40 bp of the 5&#8242; and 3&#8242; terminal sequences overlapping with 5&#8242; and 3&#8242; ends of the backbone cleavage site, respectively (Figure 1). The 5&#8242; and 3&#8242; terminal sequences of the UAS module are determined by the backbone cleavage site of the pOT2 vector.
<ol>
	<li>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 &#176;C for 1 h.</li>
	<li>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/&#181;L 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.</li>
</ol>
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).
<ol>
	<li>Test tube reaction step 1
	<ol>
		<li>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.
		<ol>
			<li>For a single reaction, add 0.16 &#181;L of 1.22 &#181;M S. pyogenes Cas9, 0.4 &#181;L of 20 ng/&#181;L sgRNA, 0.24 &#181;L of 10x Cas9 buffer, 1.2 &#181;L of DEPC-treated water. For N reactions, add (N+1) x 0.16 &#181;L of 1.22 &#181;M S. pyogenes Cas9, (N+1) x 0.4 &#181;L of 20 ng/&#181;L sgRNA, (N+1) x 0.24 &#181;L of 10x Cas9 buffer, (N+1) x 1.2 &#181;L of DEPC-treated water.</li>
		</ol>
		</li>
		<li>Vortex, mix well, and spin down. Aliquot 2 &#181;L of master mix to each tube. Add 0.4 &#181;L of 0.019 &#181;M of cDNA/ORF plasmid to each tube. Incubate cleavage reactions at 37 &#176;C for 1 h. 
		NOTE: Dilute each plasmid to 0.019 &#181;M with DEPC-treated water. If the concentration of a plasmid is lower than 0.019 &#181;M, 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.</li>
	</ol>
	</li>
	<li>Test tube reaction step 2
	<ol>
		<li>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 &#181;L single step reaction for each sample. Arrange the 0.2 mL tubes in an aluminum cooling block on ice and label the tubes carefully.
		<ol>
			<li>Prepare the master mix as follows. For a single reaction, add 0.07 &#181;L of 0.006 &#181;M UAS module and 0.7 &#181;L of single step reaction assembly master mix. For N reactions, add (N+1) x 0.07 &#181;L of 0.006 &#181;M UAS module and (N+1) x 0.7 &#181;L of single step reaction assembly master mix.</li>
		</ol>
		</li>
		<li>Vortex, mix well, and spin down. Aliquot 0.77 &#181;L of the master mix to each tube. Add 0.7 &#181;L of Cas9-linearized cDNA/ORF plasmid to each tube. Incubate reactions at 50 &#176;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.</li>
	</ol>
	</li>
	<li>Transform single step assembly reaction products into E. coli on a large scale.
	<ol>
		<li>Prechill 10 &#181;L tips at 4 &#176;C. Prechill 1.5 mL tubes in an aluminum cooling block on ice.</li>
		<li>Thaw competent E. coli cells on ice for 5 min. Aliquot 10 &#181;L 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/&#181;g of pUC19 DNA.</li>
		<li>Add 1 &#181;L of single step assembly reaction product to 10 &#181;L of competent cells, swirl gently to mix, and place on ice for 30 min.</li>
		<li>Incubate the tubes in a 42 &#176;C water bath for 1 min, and then immediately chill on ice for 2 min.</li>
		<li>Add 100 &#181;L of SOC medium (prewarmed to 37 &#176;C) into each tube at room temperature, and incubate in a shaking incubator (250 rpm) at 37 &#176;C for 1 h.</li>
		<li>Warm up to 37 &#176;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 &#176;C overnight. 
		NOTE: Performing these steps on a large scale takes several hours. Unless otherwise specified, keep reagents and reactions on ice.</li>
	</ol>
	</li>
	<li>Verify UAS-cDNA/ORF plasmids constructed by CRISPRmass
	<ol>
		<li>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 &#176;C while shaking at 250 rpm.</li>
		<li>Extract plasmid DNA by using a plasmid mini-prep kit. Set up a 20 &#181;L restriction digestion reaction as follows: 300-500 ng/&#181;L of plasmid, appropriate restriction enzyme(s), restriction digestion buffer, and ultrapure water. Incubate reactions at 37 &#176;C for 1 h.</li>
		<li>Separate DNA fragments by 0.8% agarose gel electrophoresis and analyze by a digital imaging system (Figure 3).</li>
	</ol>
	</li>
</ol>

A High-Throughput UAS-cDNA/ORF Plasmid Library Construction Using CRISPRmass

<ol><li>Adams, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genome+sequence+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The genome sequence of Drosophila melanogaster.</a> Science. 287 (5461), 2185-2195 (2000).</li>
<li>Rubin, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+genomics+of+the+eukaryotes%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Comparative genomics of the eukaryotes.</a> Science. 287 (5461), 2204-2215 (2000).</li>
<li>Reiter, L. T., Potocki, L., Chien, S., Gribskov, M., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+systematic+analysis+of+human+disease-associated+gene+sequences+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster.</a> Genome Res. 11 (6), 1114-1125 (2001).</li>
<li>Miklos, G. L., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+the+genome+project+in+determining+gene+function%3A+insights+from+model+organisms%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The role of the genome project in determining gene function: insights from model organisms.</a> Cell. 86 (4), 521-529 (1996).</li>
<li>St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+art+and+design+of+genetic+screens%3A+Drosophila+melanogaster%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">The art and design of genetic screens: Drosophila melanogaster.</a> Nat Rev Genet. 3 (3), 176-188 (2002).</li>
<li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li>
<li>Bischof, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+versatile+platform+for+creating+a+comprehensive+UAS-ORFeome+library+in+Drosophila%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila.</a> Development. 140 (11), 2434-2442 (2013).</li>
<li>Xu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+large-scale+functional+approach+to+uncover+human+genes+and+pathways+in+Drosophila%5BTitle%5D)+AND+%22Cell+Res%22%5BJournal%5D)">A large-scale functional approach to uncover human genes and pathways in Drosophila.</a> Cell Res. 18 (11), 1114-1127 (2008).</li>
<li>Wei, P., Xue, W., Zhao, Y., Ning, G., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-based+modular+assembly+of+a+UAS-cDNA%2FORF+plasmid+library+for+more+than+5500+Drosophila+genes+conserved+in+humans%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">CRISPR-based modular assembly of a UAS-cDNA/ORF plasmid library for more than 5500 Drosophila genes conserved in humans.</a> Genome Res. 30 (1), 95-106 (2020).</li>
<li>Rubin, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Drosophila+complementary+DNA+resource%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A Drosophila complementary DNA resource.</a> Science. 287 (5461), 2222-2224 (2000).</li>
<li>Stapleton, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Drosophila+gene+collection%3A+identification+of+putative+full-length+cDNAs+for+70%25+of+D.+melanogaster+genes%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes.</a> Genome Res. 12 (8), 1294-1300 (2002).</li>
<li>Yang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+public+genome-scale+lentiviral+expression+library+of+human+ORFs%5BTitle%5D)+AND+%22Nat+Meth%22%5BJournal%5D)">A public genome-scale lentiviral expression library of human ORFs.</a> Nat Meth. 8 (8), 659-661 (2011).</li>
</ol>

The authors have nothing to disclose.

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/&#181;g 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&#39; or 3&#39; end.
Unlike all the other existing methods7,8, 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&#39; end of cDNAs or ORFs. CRISPRmass promises to accelerate the development of functional genomics.

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.

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

crispr-based modular assembly for high-throughput construction of a uas-cdna/orf plasmid library

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 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.
<img alt="CRISPR plasmid assembly diagram; sgRNA targeting, cleavage, Gibson assembly, E. coli transformation." class="xfigimg" src="/files/ftp_upload/66581/66581fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
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. <a href="https://www.jove.com/files/ftp_upload/66581/66581fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="DNA electrophoresis result; pOT2-CG4968/HindIII digest; base pair ladder; band separation analysis." class="xfigimg" src="/files/ftp_upload/66581/66581fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
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. <a href="https://www.jove.com/files/ftp_upload/66581/66581fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="DNA electrophoresis results; band separation, base pairs marker; gel analysis for gene identification." class="xfigimg" src="/files/ftp_upload/66581/66581fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
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. <a href="https://www.jove.com/files/ftp_upload/66581/66581fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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. <a href="https://www.jove.com/files/ftp_upload/66581/Supplementary%20File%201_RE_v4.zip" target="_blank">Please click here to download this File.</a>

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. ...

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Research

JoVE Journal

Genetics

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Here, an automated workflow to perform modular DNA "device" assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

CRISPR-associated protein Cpf1 can be guided by a specially designed CRISPR RNA (crRNA) to cleave double-stranded DNA at desired sites, generating sticky ends. Based on this characteristic, a DNA assembly standard (C-Brick) was established, and a protocol detailing its use is described here.

Protocols for C-Brick DNA Standard Assembly Using Cpf1

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

This article presents a step-by-step protocol demonstrating how Modular Cloning (MoClo) can be adapted for the cloning of polycistronic operons.

Standardized Modular Assembly of Polycistronic Operons with Modular Cloning (MoClo) using the In-Cloning toolkit

We describe a protocol for a high-throughput cloning method, CRISPR-based shuttle cloning (CRISPRshuttle cloning), which allows the transfer of DNA fragments of interest between vectors without the need for PCR amplification of the DNA fragments.

CRISPR-based Shuttle Cloning: A High-throughput Cloning Method

Learn CRISPR-based modular assembly for high-throughput construction of UAS-cDNA/ORF plasmid libraries in Drosophila. This streamlined method enables efficient genome-wide functional genomic screening.

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

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