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><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

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 si...

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

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.

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. ...

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

Research

JoVE Journal

Genetics

847 Views.  University of South China. Functionele genomische screening biedt een krachtige benadering van de functie van sondegenen en is gebaseerd op de constructie van genoombrede plasmidebibliotheken. Conventionele benaderingen voor de bouw van plasmidebibliotheken zijn tijdrovend en arbeidsintensief. Om deze vraag te beantwoorden, ontwikkelen we een eenvoudige en efficiënte methode, CRISPR-gebaseerde modulaire assemblage, of CRISPRmass.Door splitsing van gedeelde vectorsequenties van cDNA of ORF-plasmide door CRISPR/C...

Video: Auteur in de schijnwerpers: vereenvoudiging van de constructie van genoombrede plasmidebibliotheken met behulp van CRISPRmass

Protocols for C-Brick DNA Standard Assembly Using Cpf1

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this characteristic, Cpf1 has been used for the establishment of a DNA assembly standard called C-Brick, which has the advantage of long recognition sites and short scars. On a standard C-Brick vector, there are four Cpf1 recognition sites &#8211; the prefix (T1 and T2 sites) and the suffix (T3 and T4 sites) &#8211; flanking biological DNA parts. The cleavage of T2 and T3 sites produces complementary sticky ends, which allow for the assembly of DNA parts with T2 and T3 sites. Meanwhile, a short &#34;GGATCC&#34; scar is generated between parts after assembly. As the newly formed plasmid once again contains the four Cpf1 cleavage sites, the method allows for the iterative assembly of DNA parts, which is similar to those of BioBrick and BglBrick standards. A procedure outlining the use of the C-Brick standard to assemble DNA parts is described here. The C-Brick standard can be widely used by scientists, graduate and undergraduate students, and even amateurs.

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.

CRISPR-associated protein Cpf1 cleaves double-stranded DNA under the guidance of CRISPR RNA (crRNA), generating sticky ends. Because of this ...

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

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

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.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

A Customizable Protocol for String Assembly gRNA Cloning STAgR

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.

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic ...

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

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

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.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a &#34;Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated&#34; (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

Developmental Biology

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; 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.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Bioengineering

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

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

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.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

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

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to &#946;-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

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.

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

Use of In Vivo Assembly for High-efficiency Plasmid Construction

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA fragments to assemble plasmids. This method functions by transforming DNA fragments with regions of 15-50 bp of homology into commonly used laboratory Escherichia coli strains and the bacteria use the RecA-independent repair pathway to assemble the DNA fragments into a plasmid. This method is more rapid and cost-effective than many molecular cloning methods that rely on in vitro assembly of plasmids prior to transformation into E. coli strains. This is because in vitro methods require the purchase of specialized enzymes and the performance of sequential enzymatic reactions that require incubations. However, unlike in vitro methods, IVA has not been experimentally shown to assemble linear plasmids. Here we share the IVA protocol used by our laboratory to rapidly assemble plasmids and subclone DNA fragments between plasmids with different origins of replication and antibiotic resistance markers.

In vivo assembly is a ligation-independent cloning method that relies on intrinsic DNA repair enzymes in bacteria to assemble DNA fragments by homologous recombination. This protocol is both time and cost-effective, as few reagents are required, and cloning efficiency can be as high as 99 %.

In vivo assembly (IVA) is a molecular cloning method that uses intrinsic enzymes present in bacteria that promote intermolecular recombination of DNA ...

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

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

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.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

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

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