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

Summary

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.

Abstract

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.

Introduction

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 fragments^1,2,3,4,5. 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' and 3' 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-UASC^8,9. CRISPRshuttle streamlines the construction of genome-wide plasmid libraries, thereby facilitating functional genomics studies.

Protocol

1. Determination of the optimal Cas9/sgRNA cleavage sites flanking cDNA/ORF

1. Preparation of cDNA/ORF plasmids
   1. 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.
   2. Isolate plasmid using a plasmid miniprep kit and measure its concentration with a spectrophotometer.
2. sgRNA design
   1. Access the CHOPCHOP website (https://chopchop.cbu.uib.no/). Paste the 20-100 bp region of the pLX304 vector backbone flanking the ORF 3' end into the target field.
   2. Select Drosophila melanogaster as the species and click Find Target Sites. Select sgRNA candidates that are predicted to have an efficiency of over 80%.
3. sgRNA preparation
   1. Synthesize a forward primer (5′-TAATACGACTCACTATAGG(N)₂₀GTTTTAGAGCTAGAAATAG-3′) where (N)₂₀ represents the sgRNA target sequence, and a reverse primer sgRNA-REV (5′- AAAAGCACCGACTCGGTGCCACTT-3′).
      NOTE: PAGE-purified primers are recommended.
   2. Generate a DNA template for sgRNA in vitro transcription (IVT) via PCR. Prepare a 100 µL reaction containing 5 µL each of template pX330 (Addgene plasmid 42230; 0.1 ng/µL), forward primer (10 µM), and sgRNA-REV primer (10 µM); 50 µL of 2x high-fidelity PCR master mix; and 35 µL of diethyl pyrocarbonate (DEPC)-treated ultrapure water.
   3. Amplify in a PCR Thermal Cycler using the following program: 98 °C for 30 s; 5 cycles of 98°C for 8 s, 52 °C for 15 s, 72 °C for 5 s; 30 cycles of 98 °C for 8 s, 72 °C for 20 s; 72 °C for 2 min.
   4. Resolve PCR products on a 0.8% agarose gel. Excise the ~120-bp band and purify using a gel extraction kit.
   5. Prepare a 10 µL IVT reaction containing 300 ng of purified DNA fragment, 3.33 µL of NTP buffer mix, 0.67 µL of T7 RNA polymerase mix, and DEPC-treated ultrapure water. Incubate at 37 °C for 4 h.
   6. Quantify unpurified sgRNA by spectrophotometry, dilute to 20 ng/µL with DEPC-treated ultrapure water, and freeze at -80 °C in small aliquots.
      NOTE: DNase treatment is unnecessary, as residual DNA does not interfere with downstream reactions.
4. sgRNA evaluation
   1. 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.
   2. Prepare a 5 µL Cas9 cleavage reaction mixture containing 0.2 µL of 1.22 µM S. pyogenes Cas9, 0.5 µL of 20 ng/µL sgRNA, 0.015 pmol of DNA substrate, 0.5 µL of 10x Cas9 Buffer, and DEPC-treated ultrapure water. Incubate the reaction at 37 °C for 1 h.
   3. Resolve the cleavage products using 0.8% agarose gel. Include the uncleaved DNA substrate as a negative control.
   4. Visualize the cleavage patterns using a digital imaging system and select the sgRNA with minimal residual uncleaved substrate for subsequent experiments (Figure 2).

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.

1. 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'-GTCACGACGTTGTAAAACGA-3') and Amp3-G2 (target sequence: 5'-GGAACGAAAACTCACGTTAA-3'), resulting in a 7,687 bp linear pBID-UASC vector backbone. These two sgRNAs target the 5' upstream and 3' downstream of the ampicillin resistance gene, respectively.
   1. Prepare a 10 µL cleavage reaction containing 0.03 pmol of pBID-UASC, 0.25 µL of 1.22 µM S. pyogenes Cas9, 20 ng of f1Ori5-G4, 20 ng of Amp3-G2, 1 µL 10x Cas9 Buffer, and DEPC-treated ultrapure water. Incubate at 37 °C for 1 h.
      NOTE: The Cas9-cleaved plasmid, without purification, is directly subjected to Gibson assembly.
2. 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'-TTACAATTCACTGGCC
   GTCGCGTATGTGTATGATACATAAGGTT-3') and Amp3-Cam-R (5'-AGTGGAACGAAAACTCAC
   ​GTAATTCTCATGTTTGACAGC-3') primers.
   1. Prepare the chloramphenicol resistance gene by PCR amplifying the 840 bp chloramphenicol resistance gene. Set up a 20 µL PCR reaction containing 2 µL of pMartini-Cam (0.1 ng/µL), 1 µL of f1Ori5-Cam-F (10 µM), 1 µL of Amp3-Cam-R (10 µM), 4 µL of 5x buffer, 0.4 µL of dNTPs (10 mM), 0.4 µL of high-fidelity DNA polymerase (2.5 units/µL), 11.2 µL of ultrapure water.
   2. Perform PCR using the following thermocycling conditions: 1 cycle of 95 °C for 1 min; 5 cycles of 95 °C for 15 s, 46 °C for 15 s, 72 °C for 20 s; 30 cycles of 95 °C for 15 s, 61 °C for 15 s, 72 °C for 20 s; 1 cycle of 72 °C for 2 min. Perform the reaction in a thermal cycler.
   3. Resolve the cleavage products by 0.8% agarose gel and purify an 840 bp DNA fragment using a gel extraction kit.
3. Insert the chloramphenicol resistance gene into the linearized pBID-UASC vector backbone, resulting in pBIDC-UASC.
   1. Perform a 2 µL 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 µL of 2x Gibson assembly master mix. Perform the reaction at 50 °C for 1 h.
   2. Transform 10 µL of E. coli competent cells with 1 µL of ligation reaction product. Select transformants on an LB plate supplemented with 15 µg/mL chloramphenicol.
   3. 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.

3. Construction of CRISPRshuttle-compatible destination vector

NOTE: A CRISPRshuttle cassette comprises around 20-40 bp vector backbone sequences that flank both the 5' and 3' ends of target DNA fragments, with one or two unique restriction sites located between them.

1. Anneal oligonucleotides using a thermal cycler with the following program: 94 °C for 2 min; 70 cycles of 52 s at (95-N) °C, where N represents the cycle number.
   NOTE: The annealed CRISPRshuttle cassette contains 5'-overhangs complementary to EcoRI and XbaI.
2. 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.
   1. Prepare a 5 µL ligation reaction containing 14 ng of 8,451 bp EcoRI/XbaI-digested pBIDC-UASC, 0.02 pmol of the annealed CRISPRshuttle cassette, 0.5 µL of 10x T4 DNA ligase buffer, 0.3 µL of T4 DNA ligase. Incubate the reaction overnight at 16 °C.
   2. Transform 10 µL of E. coli competent cells with 1 µL of the ligation reaction product. Select transformants on an LB plate supplemented with 15 µg/mL chloramphenicol and incubate overnight at 37 °C.
   3. 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.

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).

1. Test tube reaction step 1
   1. Excise ORFs from pLX304-ORF plasmids by cleaving the vector backbone using Cas9 in conjunction with two sgRNAs, pLX304-CMV-G1⁶ (target sequence: 5'-GAGCTCTCTGGCTAACTGTC-3', localized in the pLX304 vector backbone flanking ORF 5' end) and pLX304-3'-G1 (target sequence: 5'-TTGGTCTTAAAGTCGACGCG-3', localized in the pLX304 vector backbone flanking ORF 3' end).
      1. Prepare a master mix for a given number (N) of digestion reactions as follows: (N+1) x 0.4 µL of 1.22 µM S. pyogenes Cas9, (N+1) x 0.5 µL of 80 ng/µL pLX304-CMV-G1, (N+1) x 0.5 µL of 80 ng/µL pLX304-3'-G1, (N+1) x 0.4 µL of 10x Cas9 Buffer, and (N+1) x 1.45 µL of DEPC-treated ultrapure water.
      2. Mix thoroughly and spin down the master mix. Aliquot 3.75 µL of the master mix to each tube. Add 0.75 µL of 0.03 µM pLX304-ORF plasmid to each tube. Mix thoroughly and incubate the reactions at 37 °C for 1 h.
         NOTE: Dilute each pLX304-ORF plasmid to a concentration of 0.03 µM with DEPC-treated ultrapure water. If the concentration is less than 0.03 µM, 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.
2. Test tube reaction step 2
   1. Insert the excised ORFs into the CRISPRshuttle-compatible destination vector pBIDC-UASC-pLXvect via Gibson assembly, resulting in UAS-cDNA/ORF plasmids.
      1. 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.
      2. Prepare a master mix for a given number (N) of Gibson assembly reactions as follows: (N+1) x 0.14 µL of 3.36 µM linearized pBIDC-UASC-pLXvect, and (N+1) x 1.8 µL of Gibson assembly master mix.
      3. Mix thoroughly and spin down the master mix. Aliquot 1.94 µL of the master mix to each tube. Add 1.66 µL of Cas9-cleaved plasmid to each tube. Mix thoroughly and incubate the reactions at 50 °C for 1 h.
         NOTE: Keep the master mix and tubes ice prior to incubation at 50 °C.
3. Transform E. coli with Gibson assembly products.
   NOTE: The products of Gibson assembly do not require purification prior to E. coli transformation.
   1. Thaw bacterial competent cells on ice. Aliquot 10 µL of cells to each prechilled 1.5 mL tube.
      NOTE: Competent bacterial cells with a transformation efficiency of at least 1 × 10⁸ CFU/µg of pUC19 DNA are recommended.
   2. Gently mix 10 µL of competent cells with 1 µL of Gibson assembly products. Place on ice for 30 min.
   3. Heat-shock at 42 °C for 1 min, and then chill on ice for 2 min.
   4. Add 100 µL of prewarmed SOC medium (37 °C) into each tube. Shake at 250 rpm for 1 h at 37 °C.
   5. Place cells on LB plates containing 15 µg/mL chloramphenicol. Incubate overnight at 37 °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.
4. Verification of UAS-cDNA/ORF plasmids.
   1. Inoculate a single colony in 4.5 mL of LB medium with 15 µg/mL chloramphenicol. Shake at 250 rpm overnight at 37 °C.
   2. Isolate plasmid DNA using a plasmid mini-prep kit.
   3. Prepare a 20 µL restriction digestion reaction for each plasmid containing 300-500 ng of plasmid DNA, 0.3 µL of PvuII, 2 µL of 10x buffer, and ultrapure water. Perform the reactions at 37 °C for 1 h.
   4. Resolve DNA fragments by 0.8% agarose gel. Image under UV light (Figure 3).

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar Powder	Chembase	KBS-001H
Agarose	Sangon	A600014-0100
Automated Digital Gel Image Analysis System	Tanon	Tanon-2500B
Chloramphenicol	Sangon	A100230-0010
E.Z.N.A. Gel Extraction Kit	Omega	D2500-02
E.Z.N.A. Plasmid DNA Mini Kit I	Omega	D6942-02
EcoRI-HF	NEB	R3101S
Gibson Assembly Master Mix	NEB	E2611S
HiScribe T7 Quick High Yield RNA Synthesis Kit	NEB	E2050
NEBuilder HiFi DNA Assembly Master Mix	NEB	E2621X
PCR Thermal Cycler	LongGene	T20
Platinum SuperFi II DNA Polymerase	Thermo Scientific	12361010
PvuII-HF	NEB	R3151L
Q5 Hot Start High-Fidelity 2x Master Mix	NEB	M0494
S. pyogenes Cas9	GenScript	Z03386
Shaking Incubator	Zhichu	ZQLY-180V
Spectrophotometer	Shimadzu	BioSpec-nano
T4 DNA ligase	Promega	M1801
Trans 10 Chemically Competent Cell	TransGen	CD101-02
Tryptone	Oxoid	LP0042
XbaI	NEB	R0145S
Yeast Extract	Oxoid	LP0021