Custom Single and Double guideRNA Cloning

We provide a cloning service for custom single‑guide and double‑guide CRISPR constructs.
Design your guides using our tutorial, send us the sequences, and we will clone them into your chosen lentiviral vector.
Double‑guide constructs are now available—ideal for improving knockout efficiency or targeting two genes within the same vector.

Designing and Ordering sgRNA Oligos

Please refer to our (docx) for instructions on designing gRNA sequences using the  online tool.  Other design tools may be used, but please ensure the required overhangs and sequence elements are included for successful cloning.

Oligo ordering tips: Standard de-salted oligos (usually the most affordable option) are fully suitable for cloning. If the oligos are delivered dry, resuspend each to 100 μM in DNase‑free water. Important: Do not order oligos dissolved in TE buffer — TE will interfere with the cloning process.

If you need help with guide design or wish to outsource a complete cell‑line editing workflow, the Finnish Genome Editing Center () can assist.

Vector Backbones

We offer CRISPR/Cas9 guideRNA cloning that includes annealing your oligos and cloning them into the sgRNA scaffold of one of the following lentiviral vectors:

  • , Expresses Cas9; puromycin selection in mammalian cells.
  • , Cas9‑expressing; GFP replaces puromycin for FACS‑based selection.
  • , Cas9‑expressing; mCherry fluorescent marker; no puromycin resistance.
  • , Cas9‑expressing; blasticidin resistance.
  • , Expresses gRNA only (no Cas9); includes puromycin resistance.

Cloning to other vectors may be possible as well. Please contact us if you would like to use some other vector. 

Double-Guide Constructs

Double‑guide constructs allow:

  • increased knockout efficiency
  • simultaneous targeting of two genes

These constructs can be cloned into the same vector backbones used for single guides.

Instead of short oligos, double‑guide constructs require a DNA fragment containing:

  • guideRNA 1 + scaffold
  • a second U6 promoter
  • guideRNA 2

The first guide is expressed from the human U6 promoter.
For the second guide, you can choose between human, mouse, or synthetic U6 promoters ().

You may design the guides yourself using the instructions above, or send us your guide sequences and we will design the DNA fragment for you. The fragment is ordered from GeneArt.
Price estimate: ~65 € (April 2023, excluding VAT).
Cloning costs are the same as for single guides.

Controls

A non‑targeting single-guide control (, Sigma-Aldrich) is available for every backbone. 

Non‑gene‑targeting single‑guide and double‑guide control constructs are also available. For more information, please refer to , or feel free to contact us for additional details.

Service Includes

  • Verification of each clone by Sanger sequencing
  • A DNA miniprep of each clone
  • Bacterial glycerol stocks, which can be collected from GBU as needed

If your constructs are intended for viral delivery, we can forward them directly to HelVi‑BVC for virus production.
Acknowledging GBU in Publications

If you use a clone provided by the Genome Biology Unit (GBU) in your publication, we kindly request that you acknowledge us using the text below.

“gRNA constructs were generated by the Genome Biology Unit supported by HiLIFE, Faculty of Biological and Environmental Sciences, University of Helsinki, and Biocenter Finland"

Please also cite the creators of the vector backbone used in your construct. Citation instructions are available on the corresponding vector page on Addgene’s website.