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.
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.
|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|