SINGLE-STRANDED DNA SYNTHESIS

 

Recent research in CRISPR/Cas9 technology has shown that the use of long single-stranded DNA (ssDNA) donor templates greatly enhances the efficiency of homology directed repair (HDR) enabling researchers to optimize the process of efficiently generating transgenic animal models and cell lines.

GENEWIZ’s ssDNA synthesis service provides up to 5000 nt of full sequence-verified fragments quickly and affordably. Our fragments are derived from clonally purified double-stranded DNA (dsDNA), producing the highest quality results possible.

As the leader in working with complex gene sequences, you can trust GENEWIZ for sequence-verified ssDNA synthesis for CRISPR-mediated gene knock-in, in-vitro transcription, and much more.

Applications of ssDNA Synthesis

Antibody Discovery: Engineer customized cell lines or transgenic mouse models to study in vivo immune responses
Food technology: Modify genomes of agricultural crops to study pathogenic resistance for improved food security
Cancer Biology: Generate CRISPR gene insertions to study oncogene function for targeted therapeutics
Biofuels: Precise genome editing to optimize metabolic pathways for biofuel production

CRISPR-MEDIATED GENE KNOCK-IN WORKFLOW

Double-stranded breaks are generated through CRISPR/Cas9 editing, then repaired by the endogenous cellular pathways of non-homologous end joining (NHEJ) and HDR. While the HDR pathway has consistently proven successful in copying genetic information via homologous recombination, insertion of exogenous genetic material is a challenge due to the inherent inefficiencies of HDR. Double-stranded DNA has historically been the template of choice for gene insertions, but recent research has shown the superiority of ssODNs as a donor template for HDR. Offering much higher efficiency to insert long sequences with shorter homology arms, ssDNA has become the preferred donor template for this process. GENEWIZ now offers long (up to 5000 nt) ssDNA fragments, allowing insertion of long sequences with high efficiency and reduced cellular toxicity or off-target integration compared to dsDNA donors.

Figure 1.1. Guide RNA forms a complex with Cas9 directing enzyme to cleave target DNA resulting in a double-stranded break (DSB).

Figure 1.2. Homology directed repair after DSB in the presence of a ssDNA donor template results in precise gene knock-in.

ADVANTAGES OF ssDNA SYNTHESIS FOR CRISPR GENE KNOCK-INS

Lower cellular toxicity compared to dsDNA after cellular delivery
Low off-target integration for more reliable gene knock-ins
High specificity knock-in templates for homology directed repair
High efficiency donors for targeted insertions and gene replacements

Turnaround Time

Length Quantity (Lyophilized) Turnaround Time
151-500 nt 3µg or 10 µg or 20 µg 15-20 business days
 501-3000 nt 20-25 business days
3001-5000 nt 25-30 business days

Features & Benefits

Ph.D.-level consultation and support – Our dedicated Project Management team will tailor the order to your exact specifications and support your project from start to finish.
Advanced capabilities – GENEWIZ can synthesize sequences with difficult stretches, like highly repetitive, AT- or GC-rich DNA.
Simple online ordering – Easily place an order through your GENEWIZ account.
Quality control – Stringent quality control process with a 100% sequence accuracy guarantee.

Rapid delivery – Industry-leading turnaround time to keep your project running on time.
Yield flexibility – Choose 3, 10 or 20 µg of lyophilized fragments.


DELIVERABLES

  • Sequence verified
  • 3 µg, 10 µg, or 20 µg lyophilized DNA
  • Certificate of Analysis (COA) including gel image, sequence trace data with alignment, and sequence files

QUALITY CONTROL PROCEDURE

  • Sequence confirmation via Sanger sequencing
  • Size verification by gel electrophoresis
  • S1 nuclease digestion test

HOW TO ORDER


Place Your Order Using Our Online Ordering System


Email | Phone (1-877-GENEWIZ (436-3949), Ext. 3) | Live Chat