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 des....

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....

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