Customized CRISPR sgRNA and donor plasmids design and construction services

The type II CRISPR/Cas9 system of Cas9 from Streptococcus pyrogenes (SpCas9) can be programmed by a chimeric single-guide RNA (sgRNA), a hybrid duplex of crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) to make precise double-stranded break (DSB) in the genome. The crRNA and tracrRNA can be fused together to create a chimeric, single-guide RNA (sgRNA), and further could be construct via a sgRNA-expression plasmid. On rare occasions, certain sgRNAs may not work for reasons yet unknown; therefore, we recommend designing at least two sgRNAs for each locus and test their efficiencies in the intended cell type.

CRISPR/Cas9 is very easy to customize as this system only requires two components, the endonuclease Cas9 and a guide RNA (gRNA).

Fusion Biolabs provides customized CRISPR constructs design and construction services

  • sgRNA design and sgRNA-expression plasmid preparation
  • Targeting donor plasmid design and preparation

I. Two Vectors based CRISPR/Cas9 genome engineering system

Fusion Biolabs has simplified the design and selection of the target sequence (gRNA) and clone into our pOK1-gRNA vector (Two-vector system) and we will provide you a complimentary Cas9 expression plasmid upon request.

Single guide RNA (sgRNA) expression vector with cloning backbone for sgRNA under the promoter U6.
pOK1-gRNA expression vector (Two-vector system) with sgRNA backbone
Enhanced endonuclease activity and on-target specificity Cas9 nuclease from Streptococcus pyrogenes expression vector
pOK-eSpCas9 enhanced endonuclease activity and on-target specificity Cas9 expression vector

II. One Vector based CRISPR/Cas9 genome engineering system

Fusion Biolabs has simplified the design and selection of the target sequence (gRNA) and clone into our pOK2-gRNA vector (One-vector system).

Cas9 from S. pyogenes with 2A-Puro, and cloning backbone for sgRNA under the promoter U6
Fusion Biolabs pOK2-gRNA expression vector (One-vector system)

III. Donor plasmid construction with a predefined cassette

Site-specific genome editing tool, CRISP/Cas9 system can be customized to recognized and cleave a double-stranded DNA molecule (DSB) at a specific site, which can be subsequently repaired by endogenous cellular pathway of homology-directed repair (HDR). HDR can seamlessly repair DNA break in the presence of a DNA repair template with homologous regions spanning the cleavage site. The DNA template used in HDR can be customized by placing a GOI in between two homology arms (HAs) and then delivered exogenously in a donor plasmid.

The donor plasmid consists of the following fragments:

  1. plasmid backbone, which include elements for replication in the bacterial host;
  2. 5′ and 3′ homology arms (HA) ~750 bp each to mediate homology-directed targeted integration
  3. drug resistance cassette, harboring neomycin or other drug resistance gene together with the promoter and terminator sequences;
  4. gene expression cassette, harboring gene of interest (GOI) with the desired promoter and terminator sequences.
An overview of the donor plasmid construction. Five DNA fragments (5’ and 3’-homology arms (HAs), gene expression cassette (with GOI), drug resistance cassette (e.g., NeoR) and plasmid backbone) are assembled in the desired order and form the donor plasmid.
pOK-Donor vector for HDR gene integration (Knock-in)

Ordering Information

Customized CRISPR sgRNA and Donor plasmid Design and Construction Services

CatalogDescriptionUnitPrice
71101Target sequence gRNA cloning into pOK1-gRNA vector (2 vector system)1 ProjectContact Us
71102Target sequence gRNA cloning into pOK2-gRNA vector (1 vector system)1 ProjectContact Us
71103Donor vector design and construction with a predefined cassette1 ProjectContact Us

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