Name: CRISPR-based Shuttle Cloning: A High-throughput Cloning Method (Video) | JoVE Journal
Uploaded: 2025-06-13T13:20:00.000+00:00
Duration: 4 min 25 s
Description: Learn CRISPR-based shuttle cloning for high-throughput DNA fragment transfer between vectors without PCR amplification. This method streamlines genetic engineering workflows and reduces resource requirements.

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5 minutes

This study was supported by a grant from the National Natural Science Foundation of China (32071135) and a startup fund from the Affiliated Nanhua Hospital, Hengyang Medical School, University of South China. We are grateful to Prof. Feng Zhang for kindly providing the pX330 plasmid and to Dr. Xiaohui Cai for technical assistance.

1. Determination of the optimal Cas9/sgRNA cleavage sites flanking cDNA/ORF
<ol>
	<li>Preparation of cDNA/ORF plasmids
	<ol>
		<li>Obtain cDNA/ORF clones from public repositories. 
		NOTE: This protocol uses the pLX304 vector-based ORF clones from the human CCSB-broad Lentiviral expression library8.</li>
		<li>Isolate plasmid using a plasmid miniprep kit and measure its concentration with a spectrophotometer.</li>
	</ol>
	</li>
	<li>sgRNA design
	<ol>
		<li>Access the CHOPCHOP website (https://chopchop.cbu.uib.no/). Paste the 20-100 bp region of the pLX304 vector backbone flanking the ORF 3&#39; end into the target field.</li>
		<li>Select Drosophila melanogaster as the species and click Find Target Sites. Select sgRNA candidates that are predicted to have an efficiency of over 80%.</li>
	</ol>
	</li>
	<li>sgRNA preparation
	<ol>
		<li>Synthesize a forward primer (5&#8242;-TAATACGACTCACTATAGG(N)20GTTTTAGAGCTAGAAATAG-3&#8242;) where (N)20 represents the sgRNA target sequence, and a reverse primer sgRNA-REV (5&#8242;- AAAAGCACCGACTCGGTGCCACTT-3&#8242;). 
		NOTE: PAGE-purified primers are recommended.</li>
		<li>Generate a DNA template for sgRNA in vitro transcription (IVT) via PCR. Prepare a 100 &#181;L reaction containing 5 &#181;L each of template pX330 (Addgene plasmid 42230; 0.1 ng/&#181;L), forward primer (10 &#181;M), and sgRNA-REV primer (10 &#181;M); 50 &#181;L of 2x high-fidelity PCR master mix; and 35 &#181;L of diethyl pyrocarbonate (DEPC)-treated ultrapure water.</li>
		<li>Amplify in a PCR Thermal Cycler using the following program: 98 &#176;C for 30 s; 5 cycles of 98&#176;C for 8 s, 52 &#176;C for 15 s, 72 &#176;C for 5 s; 30 cycles of 98 &#176;C for 8 s, 72 &#176;C for 20 s; 72 &#176;C for 2 min.</li>
		<li>Resolve PCR products on a 0.8% agarose gel. Excise the ~120-bp band and purify using a gel extraction kit.</li>
		<li>Prepare a 10 &#181;L IVT reaction containing 300 ng of purified DNA fragment, 3.33 &#181;L of NTP buffer mix, 0.67 &#181;L of T7 RNA polymerase mix, and DEPC-treated ultrapure water. Incubate at 37 &#176;C for 4 h.</li>
		<li>Quantify unpurified sgRNA by spectrophotometry, dilute to 20 ng/&#181;L with DEPC-treated ultrapure water, and freeze at -80 &#176;C in small aliquots. 
		NOTE: DNase treatment is unnecessary, as residual DNA does not interfere with downstream reactions.</li>
	</ol>
	</li>
	<li>sgRNA evaluation
	<ol>
		<li>Digest a cDNA/ORF plasmid containing the pLX304 vector backbone using a restriction enzyme. Resolve the resulting DNA fragments via 0.8% agarose gel. Purify the DNA substrate using a gel extraction kit.</li>
		<li>Prepare a 5 &#181;L Cas9 cleavage reaction mixture containing 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 ultrapure water. Incubate the reaction at 37 &#176;C for 1 h.</li>
		<li>Resolve the cleavage products using 0.8% agarose gel. Include the uncleaved DNA substrate as a negative control.</li>
		<li>Visualize the cleavage patterns using a digital imaging system and select the sgRNA with minimal residual uncleaved substrate for subsequent experiments (Figure 2).</li>
	</ol>
	</li>
</ol>
2. Swapping the antibiotic resistance gene of the destination vector
NOTE: In this protocol, we use the example of swapping the ampicillin resistance gene of the destination vector pBID-UASC with the chloramphenicol resistance gene, thereby generating the chloramphenicol-bearing destination vector pBIDC-UASC.
<ol>
	<li>Remove the ampicillin resistance gene from pBID-UASC. 
	NOTE: The ampicillin resistance gene of pBID-UASC was removed by Cas9 digestion in conjunction with two sgRNAs, f1Ori5-G4 (target sequence: 5&#39;-GTCACGACGTTGTAAAACGA-3&#39;) and Amp3-G2 (target sequence: 5&#39;-GGAACGAAAACTCACGTTAA-3&#39;), resulting in a 7,687 bp linear pBID-UASC vector backbone. These two sgRNAs target the 5&#39; upstream and 3&#39; downstream of the ampicillin resistance gene, respectively.
	<ol>
		<li>Prepare a 10 &#181;L cleavage reaction containing 0.03 pmol of pBID-UASC, 0.25 &#181;L of 1.22 &#181;M S. pyogenes Cas9, 20 ng of f1Ori5-G4, 20 ng of Amp3-G2, 1 &#181;L 10x Cas9 Buffer, and DEPC-treated ultrapure water. Incubate at 37 &#176;C for 1 h. 
		NOTE: The Cas9-cleaved plasmid, without purification, is directly subjected to Gibson assembly.</li>
	</ol>
	</li>
	<li>PCR-amplify the chloramphenicol resistance gene. 
	NOTE: An 840 bp chloramphenicol resistance gene was amplified from the plasmid pMartini-Cam6 by PCR using the f1Ori5-Cam-F (5&#39;-TTACAATTCACTGGCC 
	GTCGCGTATGTGTATGATACATAAGGTT-3&#39;) and Amp3-Cam-R (5&#39;-AGTGGAACGAAAACTCAC 
	&#8203;GTAATTCTCATGTTTGACAGC-3&#39;) primers.
	<ol>
		<li>Prepare the chloramphenicol resistance gene by PCR amplifying the 840 bp chloramphenicol resistance gene. Set up a 20 &#181;L PCR reaction containing 2 &#181;L of pMartini-Cam (0.1 ng/&#181;L), 1 &#181;L of f1Ori5-Cam-F (10 &#181;M), 1 &#181;L of Amp3-Cam-R (10 &#181;M), 4 &#181;L of 5x buffer, 0.4 &#181;L of dNTPs (10 mM), 0.4 &#181;L of high-fidelity DNA polymerase (2.5 units/&#181;L), 11.2 &#181;L of ultrapure water.</li>
		<li>Perform PCR using the following thermocycling conditions: 1 cycle of 95 &#176;C for 1 min; 5 cycles of 95 &#176;C for 15 s, 46 &#176;C for 15 s, 72 &#176;C for 20 s; 30 cycles of 95 &#176;C for 15 s, 61 &#176;C for 15 s, 72 &#176;C for 20 s; 1 cycle of 72 &#176;C for 2 min. Perform the reaction in a thermal cycler.</li>
		<li>Resolve the cleavage products by 0.8% agarose gel and purify an 840 bp DNA fragment using a gel extraction kit.</li>
	</ol>
	</li>
	<li>Insert the chloramphenicol resistance gene into the linearized pBID-UASC vector backbone, resulting in pBIDC-UASC.
	<ol>
		<li>Perform a 2 &#181;L Gibson assembly reaction: 0.008 pmol of 840 bp chloramphenicol resistance gene, 0.002 pmol of the linearized pBID-UASC (step 2.1.1), 1.0 &#181;L of 2x Gibson assembly master mix. Perform the reaction at 50 &#176;C for 1 h.</li>
		<li>Transform 10 &#181;L of E. coli competent cells with 1 &#181;L of ligation reaction product. Select transformants on an LB plate supplemented with 15 &#181;g/mL chloramphenicol.</li>
		<li>Pick a single colony and subject it to subsequent bacterial culture and plasmid miniprep. Verify the chloramphenicol-bearing destination vector pBIDC-UASC by restriction analysis and DNA sequencing.</li>
	</ol>
	</li>
</ol>
3. Construction of CRISPRshuttle-compatible destination vector
NOTE: A CRISPRshuttle cassette comprises around 20-40 bp vector backbone sequences that flank both the 5&#39; and 3&#39; ends of target DNA fragments, with one or two unique restriction sites located between them.
<ol>
	<li>Anneal oligonucleotides using a thermal cycler with the following program: 94 &#176;C for 2 min; 70 cycles of 52 s at (95-N) &#176;C, where N represents the cycle number. 
	NOTE: The annealed CRISPRshuttle cassette contains 5&#39;-overhangs complementary to EcoRI and XbaI.</li>
	<li>Ligate the annealed CRISPRshuttle cassette into EcoRI/XbaI-digested destination vector pBIDC-UASC to generate the CRISPRshuttle-compatible destination vector pBIDC-UASC-pLXvect. 
	NOTE: This ligation eliminates the original EcoRI and XbaI sites within the multiple cloning sites, rendering the EcoRI and XhoI sites in the middle of the CRISPRshuttle cassette unique in the final vector. The unique restriction sites in the CRISPRshuttle cassette enable linearization of the CRISPRshuttle-compatible destination vector.
	<ol>
		<li>Prepare a 5 &#181;L ligation reaction containing 14 ng of 8,451 bp EcoRI/XbaI-digested pBIDC-UASC, 0.02 pmol of the annealed CRISPRshuttle cassette, 0.5 &#181;L of 10x T4 DNA ligase buffer, 0.3 &#181;L of T4 DNA ligase. Incubate the reaction overnight at 16 &#176;C.</li>
		<li>Transform 10 &#181;L of E. coli competent cells with 1 &#181;L of the ligation reaction product. Select transformants on an LB plate supplemented with 15 &#181;g/mL chloramphenicol and incubate overnight at 37 &#176;C.</li>
		<li>Pick a single colony and subject it to subsequent bacterial culture and plasmid miniprep. Verify the CRISPRshuttle-compatible destination vector pBIDC-UASC-pLXvect by restriction analysis and DNA sequencing.</li>
	</ol>
	</li>
</ol>
4. Generation of UAS-cDNA/ORF plasmids using CRISPRshuttle
NOTE: The CRISPRshuttle protocol involves two-step test tube reactions being carried out in parallel before bacterial transformation (Figure 1).
<ol>
	<li>Test tube reaction step 1
	<ol>
		<li>Excise ORFs from pLX304-ORF plasmids by cleaving the vector backbone using Cas9 in conjunction with two sgRNAs, pLX304-CMV-G16 (target sequence: 5&#39;-GAGCTCTCTGGCTAACTGTC-3&#39;, localized in the pLX304 vector backbone flanking ORF 5&#39; end) and pLX304-3&#39;-G1 (target sequence: 5&#39;-TTGGTCTTAAAGTCGACGCG-3&#39;, localized in the pLX304 vector backbone flanking ORF 3&#39; end).
		<ol>
			<li>Prepare a master mix for a given number (N) of digestion reactions as follows: (N+1) x 0.4 &#181;L of 1.22 &#181;M S. pyogenes Cas9, (N+1) x 0.5 &#181;L of 80 ng/&#181;L pLX304-CMV-G1, (N+1) x 0.5 &#181;L of 80 ng/&#181;L pLX304-3&#39;-G1, (N+1) x 0.4 &#181;L of 10x Cas9 Buffer, and (N+1) x 1.45 &#181;L of DEPC-treated ultrapure water.</li>
			<li>Mix thoroughly and spin down the master mix. Aliquot 3.75 &#181;L of the master mix to each tube. Add 0.75 &#181;L of 0.03 &#181;M pLX304-ORF plasmid to each tube. Mix thoroughly and incubate the reactions at 37 &#176;C for 1 h. 
			NOTE: Dilute each pLX304-ORF plasmid to a concentration of 0.03 &#181;M with DEPC-treated ultrapure water. If the concentration is less than 0.03 &#181;M, add plasmid DNA directly to the reaction. The Cas9-cleaved plasmids do not need to be purified after cleavage reactions and can be directly used for subsequent Gibson assembly.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Test tube reaction step 2
	<ol>
		<li>Insert the excised ORFs into the CRISPRshuttle-compatible destination vector pBIDC-UASC-pLXvect via Gibson assembly, resulting in UAS-cDNA/ORF plasmids.
		<ol>
			<li>Digest pBIDC-UASC-pLXvect with EcoRI and XbaI. Separate the linearized pBIDC-UASC-pLXvect by agarose gel electrophoresis and purify using a gel extraction kit.</li>
			<li>Prepare a master mix for a given number (N) of Gibson assembly reactions as follows: (N+1) x 0.14 &#181;L of 3.36 &#181;M linearized pBIDC-UASC-pLXvect, and (N+1) x 1.8 &#181;L of Gibson assembly master mix.</li>
			<li>Mix thoroughly and spin down the master mix. Aliquot 1.94 &#181;L of the master mix to each tube. Add 1.66 &#181;L of Cas9-cleaved plasmid to each tube. Mix thoroughly and incubate the reactions at 50 &#176;C for 1 h. 
			NOTE: Keep the master mix and tubes ice prior to incubation at 50 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transform E. coli with Gibson assembly products. 
	NOTE: The products of Gibson assembly do not require purification prior to E. coli transformation.
	<ol>
		<li>Thaw bacterial competent cells on ice. Aliquot 10 &#181;L of cells to each prechilled 1.5 mL tube. 
		NOTE: Competent bacterial cells with a transformation efficiency of at least 1 &#215; 108 CFU/&#181;g of pUC19 DNA are recommended.</li>
		<li>Gently mix 10 &#181;L of competent cells with 1 &#181;L of Gibson assembly products. Place on ice for 30 min.</li>
		<li>Heat-shock at 42 &#176;C for 1 min, and then chill on ice for 2 min.</li>
		<li>Add 100 &#181;L of prewarmed SOC medium (37 &#176;C) into each tube. Shake at 250 rpm for 1 h at 37 &#176;C.</li>
		<li>Place cells on LB plates containing 15 &#181;g/mL chloramphenicol. Incubate overnight at 37 &#176;C. 
		NOTE: Conducting these procedures on a large scale typically requires several hours. Unless stated otherwise, keep both the reagents and the reaction set-ups on ice.</li>
	</ol>
	</li>
	<li>Verification of UAS-cDNA/ORF plasmids.
	<ol>
		<li>Inoculate a single colony in 4.5 mL of LB medium with 15 &#181;g/mL chloramphenicol. Shake at 250 rpm overnight at 37 &#176;C.</li>
		<li>Isolate plasmid DNA using a plasmid mini-prep kit.</li>
		<li>Prepare a 20 &#181;L restriction digestion reaction for each plasmid containing 300-500 ng of plasmid DNA, 0.3 &#181;L of PvuII, 2 &#181;L of 10x buffer, and ultrapure water. Perform the reactions at 37 &#176;C for 1 h.</li>
		<li>Resolve DNA fragments by 0.8% agarose gel. Image under UV light (Figure 3).</li>
	</ol>
	</li>
</ol>

<ol><li>Hartley, J. L., Temple, G. F., Brasch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+cloning+using+in+vitro+site-specific+recombination%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">DNA cloning using in vitro site-specific recombination.</a> Genome Res. 10 (11), 1788-1795 (2000).</li>
<li>Brasch, M. A., Hartley, J. L., Vidal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orfeome+cloning+and+systems+biology%3A+Standardized+mass+production+of+the+parts+from+the+parts-list%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Orfeome cloning and systems biology: Standardized mass production of the parts from the parts-list.</a> Genome Res. 14 (10B), 2001-2009 (2004).</li>
<li>Zhu, B., Cai, G., Hall, E. O., Freeman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In-fusion+assembly%3A+Seamless+engineering+of+multidomain+fusion+proteins%2C+modular+vectors%2C+and+mutations%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">In-fusion assembly: Seamless engineering of multidomain fusion proteins, modular vectors, and mutations.</a> Biotechniques. 43 (3), 354-359 (2007).</li>
<li>Liu, Q., Li, M. Z., Leibham, D., Cortez, D., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+univector+plasmid-fusion+system%2C+a+method+for+rapid+construction+of+recombinant+DNA+without+restriction+enzymes%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes.</a> Curr Biol. 8 (24), 1300-1309 (1998).</li>
<li>Marsischky, G., Labaer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Many+paths+to+many+clones%3A+A+comparative+look+at+high-throughput+cloning+methods%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Many paths to many clones: A comparative look at high-throughput cloning methods.</a> Genome Res. 14 (10b), 2020-2028 (2004).</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>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR-based+shuttle+cloning+of+1%2C397+human+genes+into+UAS+vectors%5BTitle%5D)+AND+%22Acta+Biochim+Biophys+Sin+%28Shanghai%29%22%5BJournal%5D)">CRISPR-based shuttle cloning of 1,397 human genes into UAS vectors.</a> Acta Biochim Biophys Sin (Shanghai). , (2025).</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+Methods%22%5BJournal%5D)">A public genome-scale lentiviral expression library of human ORFs.</a> Nat Methods. 8 (8), 659-661 (2011).</li>
<li>Wang, J. W., Beck, E. S., Mccabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+modular+toolset+for+recombination+transgenesis+and+neurogenetic+analysis+of+Drosophila%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A modular toolset for recombination transgenesis and neurogenetic analysis of Drosophila.</a> PLoS One. 7 (7), e42102(2012).</li>
<li>Christie, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precise+DNA+cleavage+using+CRISPR-SpRYgests%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Precise DNA cleavage using CRISPR-SpRYgests.</a> Nat Biotechnol. 41 (3), 409-416 (2023).</li>
<li>Li, S. Y., Zhao, G. P., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C-brick%3A+A+new+standard+for+assembly+of+biological+parts+using+Cpf1%5BTitle%5D)+AND+%22ACS+Synth+Biol%22%5BJournal%5D)">C-brick: A new standard for assembly of biological parts using Cpf1.</a> ACS Synth Biol. 5 (12), 1383-1388 (2016).</li>
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</ol>

The authors have no conflicts of interest to disclose.

A critical step in the CRISPRshuttle protocol is the preparation of linearized CRISPRshuttle-compatible destination vectors. To ensure complete digestion, use an excess of restriction enzymes to digest the vectors, and gel purification of the digested vectors is strongly recommended. Another crucial step is the digestion of cDNA/ORF plasmids with Cas9. If plasmid construction fails, use agarose gel electrophoresis to check whether at least partial cDNAs/ORFs have been released from the donor plasmids. Alternatively, chec...

Constructing genome-wide plasmid libraries from available resources is the foundation and prerequisite for employing functional genomics to dissect biological processes. Current high-throughput cloning methods, including Gateway, In-Fusion, Creator, and Univector cloning systems, necessitate PCR amplification of target DNA fragments1,2,3,4,5. This prerequisite entails fragment-specific processing workflows encompassing multiple standardized operations, including but not limited to oligonucleotide primer design, gel purification, and sequence validation through sequencing. As a result, constructing genome-wide plasmid libraries (e.g., cDNA/ORF overexpression libraries) has become labor-intensive and time-consuming, impeding the advancement of functional genomics.
Previously, we developed CRISPRmass, a high-throughput cloning method designed to integrate specific DNA fragments (e.g., the UAS module) into multiple plasmids sharing identical vector backbones6. Using CRISPRmass, we constructed more than 5,500 GAL4/UAS-based UAS-cDNA/ORF plasmids from a Drosophila cDNA/ORF library, the Drosophila Genomics Resource center (DGRC) Gold Collection6. However, CRISPRmass lacks the capability to transfer DNA fragments between vectors, restricting its application in high-throughput cloning.
To address these limitations, we developed CRISPR-based shuttle cloning (CRISPRshuttle), a novel high-throughput method that facilitates the transfer of multiple target DNA fragments to destination vectors from donor plasmids7. This process requires only two sequential test-tube reactions, thereby circumventing the requirement for fragment-specific handling of discrete DNA targets7.
The CRISPRshuttle protocol involves two sequential test tube reactions (Figure 1). First, shared vector backbone sequences of donor plasmids are cleaved by Cas9/sgRNA to release target DNA fragments. These fragments are then transferred to the CRISPRshuttle cassette of a CRISPRshuttle-compatible vector via Gibson assembly to generate the final plasmids. A CRISPRshuttle cassette comprises a ~20-40 bp vector backbone sequence that flanks the 5&#39; and 3&#39; ends of the DNA fragments originating from donor plasmids, and one or two unique restriction enzyme recognition sites located between these flanking sequences. A CRISPRshuttle-compatible vector is constructed by inserting a CRISPRshuttle cassette into a destination vector, which is subsequently linearized by digesting the restriction sites within the cassette. The destination vector must carry an antibiotic resistance gene distinct from those in donor plasmids; if identical, the resistance gene must be replaced with a distinct one prior to use.
Here, we present a detailed protocol utilizing CRISPRshuttle to build a UAS-cDNA/ORF plasmid library. This process involves transferring human ORFs from the CCSB-Broad Lentiviral Expression Library into the Drosophila transgenesis vector pBID-UASC8,9. CRISPRshuttle streamlines the construction of genome-wide plasmid libraries, thereby facilitating functional genomics studies.

<table><tbody><tr><td>Agar Powder</td><td>Chembase</td><td>KBS-001H</td><td></td></tr><tr><td>Agarose</td><td>Sangon</td><td>A600014-0100</td><td></td></tr><tr><td>Automated Digital Gel Image Analysis System</td><td>Tanon</td><td>Tanon-2500B</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sangon</td><td>A100230-0010</td><td></td></tr><tr><td>E.Z.N.A. Gel Extraction Kit</td><td>Omega</td><td>D2500-02</td><td></td></tr><tr><td>E.Z.N.A. Plasmid DNA Mini Kit I</td><td>Omega</td><td>D6942-02</td><td></td></tr><tr><td>EcoRI-HF</td><td>NEB</td><td>R3101S</td><td></td></tr><tr><td>Gibson Assembly Master Mix</td><td>NEB</td><td>E2611S</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>E2621X</td><td></td></tr><tr><td>PCR Thermal Cycler</td><td>LongGene</td><td>T20</td><td></td></tr><tr><td>Platinum SuperFi II DNA Polymerase</td><td>Thermo Scientific</td><td>12361010</td><td></td></tr><tr><td>PvuII-HF</td><td>NEB</td><td>R3151L</td><td></td></tr><tr><td>Q5 Hot Start High-Fidelity 2x Master Mix</td><td>NEB</td><td>M0494</td><td></td></tr><tr><td>&lt;em&gt;S. pyogenes&lt;/em&gt; Cas9</td><td>GenScript</td><td>Z03386</td><td></td></tr><tr><td>Shaking Incubator</td><td>Zhichu</td><td>ZQLY-180V</td><td></td></tr><tr><td>Spectrophotometer</td><td>Shimadzu</td><td>BioSpec-nano</td><td></td></tr><tr><td>T4 DNA ligase</td><td>Promega</td><td>M1801</td><td></td></tr><tr><td>&lt;em&gt;Trans &lt;/em&gt;10 Chemically Competent Cell</td><td>TransGen</td><td>CD101-02</td><td></td></tr><tr><td>Tryptone</td><td>Oxoid</td><td>LP0042</td><td></td></tr><tr><td>XbaI</td><td>NEB</td><td>R0145S</td><td></td></tr><tr><td>Yeast Extract</td><td>Oxoid</td><td>LP0021</td><td></td></tr></tbody></table>

crispr-based shuttle cloning: a high-throughput cloning method

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

The development of genome-wide plasmid libraries using existing genomic repositories serves as a pivotal prerequisite for systematic functional characterization of genes across diverse biological processes. Current high-throughput methodologies for inter-vector DNA fragment transfer, however, necessitate PCR amplification of target sequences prior to cloning, rendering the generation of genome-scale plasmid collections technically demanding and time-intensive. By leveraging a CRISPRshuttle cassette, we developed a new high-throughput cloning method, CRISPR-based shuttle cloning (CRISPRshuttle cloning), which facilitates the transfer of many DNA fragments from donor plasmids sharing identical backbone sequences to a CRISPRshuttle-compatible vector without PCR amplification of the DNA fragments. Here, we present a protocol for CRISPRshuttle. This protocol involves two sequential test tube reactions prior to bacterial transformation. First, target DNA fragments are excised from donor plasmids by Cas9-mediated cleavage of their shared vector backbone sequence. Second, the excised DNA fragments are inserted into linearized CRISPRshuttle-compatible vectors through Gibson assembly. Our results demonstrate that the efficiency of CRISPRshuttle exceeds 94% and that two researchers can generate about 300 plasmids in 7 days using CRISPRshuttle. CRISPRshuttle facilitates efficient, adaptable, and cost-effective DNA fragment transfer between vectors, significantly streamlining genome-wide plasmid library generation.

We utilized CRISPRshuttle to construct a UAS-cDNA/ORF plasmid collection covering 1,397 human genes that are conserved in Drosophila7. Restriction analysis revealed that CRISPRshuttle reaches an efficiency of 94.5% for using CRISPRshuttle-compatible destination vectors containing two repetitive sequences and 96.1% for using destination vectors without repetitive sequences7. Our data demonstrated that generally ~300 plasmids can be created via CRISPRshuttle by two r...

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.

The development of genome-wide plasmid libraries using existing genomic repositories serves as a pivotal prerequisite for systematic functional ...

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Research

JoVE Journal

Biochemistry

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles.  Here, we describe an improved method of recombineering in E. coli, that we term ‘subcloning plus insertion’, which can be used to generate custom gene targeting vectors rapidly.

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

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

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined.

CRISPR Guide RNA Cloning for Mammalian Systems

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

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

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

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

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

The THP-1 cell line is widely used as a model to investigate the functions of human monocytes/macrophages across various biology-related research areas. This article describes a protocol for efficient CRISPR-Cas9-based engineering and single-cell clone isolation, enabling the production of robust and reproducible phenotypic data.

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

Learn CRISPR-based shuttle cloning for high-throughput DNA fragment transfer between vectors without PCR amplification. This method streamlines genetic engineering workflows and reduces resource requirements.

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

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