One-step Overlapping PCR for Rapid Synthesis of Single-guide RNA DNA Templates for the CRISPR System

Summary

A one-step overlapping PCR method using four primers enables rapid, low-cost generation of DNA templates for in vitro sgRNA transcription. An optimized primer ratio (50:5:1:50) produces high-quality sgRNAs within 5 h, with functional validation demonstrated through in vitro assays and editing activity in HEK293T cells.

Abstract

The CRISPR–Cas system has revolutionized genome editing; however, conventional methods for generating single-guide RNA (sgRNA) often involve time-consuming cloning steps or expensive commercial synthesis kits. An optimized one-step overlapping PCR strategy is presented for the rapid, cost-effective synthesis of DNA templates for in vitro sgRNA transcription. Using four partially overlapping primers spanning the T7 promoter, target-specific guide sequence, and sgRNA scaffold, full-length templates are assembled in a single PCR reaction without cloning. Systematic experimental optimization established an optimal primer ratio (AF1:AF2:AF3:Tracr-R = 50:5:1:50), minimizing non-specific byproducts while maximizing full-length product yield, as confirmed by agarose gel electrophoresis. This approach was successfully extended to generate templates for Staphylococcus aureus Cas9 (saCas9) sgRNA, demonstrating cross-system applicability beyond Streptococcus pyogenes Cas9 (SpCas9). Although direct chemical synthesis of sgRNAs offers advantages such as high purity, chemical modifications to enhance stability, and reduced off-target effects, it remains prohibitively expensive for high-throughput applications or large-scale screens that require numerous sgRNAs. In vitro cleavage assays demonstrated that guide RNAs generated using this method achieve editing efficiencies comparable to those obtained via conventional plasmid-based cloning. Furthermore, ribonucleoprotein complexes assembled with these sgRNAs and delivered into HEK293T cells via electroporation resulted in detectable indel formation at the target locus, confirming functionality in vivo. Cost analysis indicates that this method substantially reduces template preparation costs compared to commercial synthesis kits while reducing turnaround time from days to hours, thereby providing an accessible and scalable approach for laboratories engaged in genetic research.

Introduction

The CRISPR–Cas system, derived from an adaptive immune mechanism in bacteria and archaea, has revolutionized genetic engineering by enabling precise genome editing across diverse organisms^1,2. A key component is the single-guide RNA (sgRNA), a chimeric RNA that directs Cas nucleases to specific genomic loci; its modular design allows retargeting through modification of an approximately 20-nucleotide protospacer sequence³. Beyond SpCas9, other Cas nucleases with distinct properties have been widely adopted. Staphylococcus aureus Cas9 (saCas9) is smaller, facilitating adeno-associat....

Protocol

The use of the commercially available HEK293T cell line (ATCC CRL-3216) was conducted in accordance with the Ethical Committee of the First People's Hospital of Chenzhou City, University of South China guidelines for the use of human-derived cell lines. The reagents, software, and equipment used are listed in the Table of Materials.

1. Design of sgRNA target sequences

1. Identify the target genomic region using established online tools (e.g., CRISPRscan, CHOPCHOP, or Broad Institute CRISPick)³.
2. Access the CHOPCHOP web server at https://chopchop.cbu.uib.no/

Results

Successful application of the one-step overlapping PCR protocol produces a single, sharp DNA band of the expected size when analyzed by agarose gel electrophoresis.

Suboptimal primer ratios, particularly those with excessive concentrations of inner primers (AF2 and AF3), result in additional bands larger than the expected product, indicating non-specific amplification or primer-dimer formation, along with faint bands corresponding to incomplete assembly intermediates (Figu.......

Discussion

A streamlined, economical protocol for synthesizing sgRNA DNA templates via a one-step overlapping PCR strategy is described. This method eliminates the need for plasmid construction and cloning, reducing template preparation time from several days to approximately 5 h. A key aspect of this approach is the systematic optimization of the primer ratio, determined to be 50:5:1:50 (AF1:AF2:AF3:Tracr-R), corresponding to working concentrations of 10 µM, 1 µM, 0.2 µM, and 10 µM, respectively. This .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 bp DNA Ladder	TIANGEN	MD109	DNA size marker for electrophoresis
2Taq Plus MasterMix	CWBIO	CW2849M	PCR amplification reagent
50 bp DNA Ladder	TIANGEN	MD108	DNA size marker for small fragments
6DNA loading buffer	TIANGEN	1109565	Sample loading buffer for electrophoresis
Agarose Gel DNA Recovery Kit	TIANGEN	DP219	DNA purification from agarose gel
Agarose I	Thermo Scientific	17850	Agarose gel preparation
Broad Institute CRISPick	https://portals.broadinstitute.org/gppx/crispick/public		sgRNA design tool
Centrifuge	HENGNUO	2-16R	Sample centrifugation
Certified Copper Fetal Bovine Serum	Umedium, Beijing BioTopped Technology Co., Ltd	3023A	Cell culture supplement
CH3CO2H	Shanghai Aladdin Biochemical Technology Co., Ltd	A758682	TAE buffer solution preparation reagents
CHOPCHOP	https://chopchop.cbu.uib.no/		sgRNA design tool
CO2 incubator	Thermo Scientific	51032874	Mammalian cell culture incubation
CRISPRscan	https://www.crisprscan.org/		sgRNA design tool
DMEM (Dulbecco's Modified Eagle Medium)	KeyGEN BioTECH	KGL1211-500	Cell culture medium
dsDNase	Thermo Scientific	EN0771	Removal of DNA template after transcription
ECM 830 Square Wave Electroporation System	BTX	ECM830	Electroporation of cells
EDTA Na2	BioSharp	BS108	TAE buffer solution preparation reagents
Electric thermostatic water bath	SHANGHAI SUMSUNG LABORATORY INSTRUMENT CO.,LTD	DKD8	Temperature-controlled incubation
Electrophoresis Apparatus Trophoresis	LIUYI	DYCP-31BN	Agarose gel electrophoresis
Gel Imaging analysis system	ShanghHai JiaPeng	ZF-258	Gel visualization and imaging
GenCRISPR SpCas9	GenScript	Z03624-GMP-2.5	Cas9 nuclease for genome editing
Genomic DNA extraction kit	TIANGEN	YDP304	Genomic DNA isolation
HiScribe T7 High Yield RNA Synthesis Kit	NEB	E2040S	In vitro transcription of sgRNA
MiniAmp Thermocycler	Thermo Scientific	A37834	PCR amplification
NanoDrop 2000c Spectrophotometer	Thermo Scientific	2000c	Nucleic acid quantification
Nucleic acid stain (e.g., GeneRed)	TIANGEN	RT211	DNA visualization in agarose gel
PCR Using Q5 Quick-Load High-Fidelity 2X Master Mix	NEB	M0578	High-fidelity PCR amplification
Primers (AF1, AF2, AF3, Tracr-R)	GenScript		Oligonucleotides for sgRNA template assembly
Tris-base	BioSharp	BS083	TAE buffer solution preparation reagents
VAHTS RNA Clean Beads	VAHTS	N412-01	RNA purification