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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+Drosophila+melanogaster.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomics+of+the+eukaryotes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+analysis+of+human+disease-associated+gene+sequences+in+Drosophila+melanogaster.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+genome+project+in+determining+gene+function:+insights+from+model+organisms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+art+and+design+of+genetic+screens:+Drosophila+melanogaster.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+platform+for+creating+a+comprehensive+UAS-ORFeome+library+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+functional+approach+to+uncover+human+genes+and+pathways+in+Drosophila.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-based+modular+assembly+of+a+UAS-cDNA/ORF+plasmid+library+for+more+than+5500+Drosophila+genes+conserved+in+humans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+complementary+DNA+resource.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+gene+collection:+identification+of+putative+full-length+cDNAs+for+70%+of+D.+melanogaster+genes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+public+genome-scale+lentiviral+expression+library+of+human+ORFs.">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 digest...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</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

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;

Author Spotlight: Simplifying Genome-Wide Plasmid Library Construction Using CRISPRmass

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

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.

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 si...

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. ...

crispr-based-modular-assembly-for-high-throughput-construction-uas

Research

JoVE Journal

Genetics

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

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.

500 Views.  University of South China.  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.

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

Summary