Single and double custom guide cloning

Design your own guides with our tutorial and we'll clone them into lentivectors for you.

New! Double-guide constructs are now available! Make your knock out more efficient with double guides or target two genes in one construct.
gRNA cloning service

We now offer a CRISPR/Cas9 cloning service that includes the annealing and cloning of your custom oligos into the single guide RNA scaffold of lentiviral vectors. You can choose between four different lentivectors:

lentiCRISPRv2, which allows the delivery of the desired single guide RNA (sgRNA) and Cas9 nuclease in mammalian cells, and selection with puromycin
lentiCRISPRv2GFP, which has Cas9 but puromycin resistance is replaced by GFP for selection with FACS
lentiCRISPRv2-mCherry, which has Cas9 and mCherry fluorescent tag but no puromycin resistance
lentiGuide-Puro, which allows the expression of the chimeric guide RNA only. It does not contain Cas9 but contains puromycin resistance. 

Controls: Non-targeting control constructs are available in every backbone with same gRNA sequence as CRISPR19 control in the Sanger library.

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


Guide design

Please refer to our step-by-step guide (docx) on how to design gRNA sequences using the Benchling online program as a tool. It is also possible to use a different tool to design the guides, but keep in mind that there are specific sequences to incorporate in the oligo for the cloning to work.

Oligonucleotide ordering tips: Standard de-salted oligos (usually the most inexpensive synthesis) are sufficient for cloning. If not already resuspended, dilute each oligo to 100 μM in DNAse-free water.

Double-guide constructs

Make your knock out more efficient with double guides or target two genes in one construct. The double-guide constructs can be cloned into the same backbones as the single guides. Instead of short oligos, we order a PCR fragment containing the guide1 and scaffold, another U6 promoter, and guide2. The PCR fragment is ordered from GeneArt and the price of the construct is around 65 € (April 2023, not including VAT). The price for the cloning is the same as for the single guides.

The first guide will be expressed by the human U6 promoter. You can choose the promoter for the second guide from human, mouse, and synthetic (Vidigal & Ventura, 2015)  U6 promoters. You can use the instructions above to design the guides or use any guide sequences you wish. Send us your guide sequences and we will design the fragment for you. 


New prices starting 1.3.2024:

Customer class University of Helsinki Biocenter finland external
gRNA cloning      
1st item in batch 100 € 115 € 475 €
Additional items 60 € 70 € 165 €

A batch is defined as samples of one customer processed at the same time. Control constructs cost 20 € /miniprep. 


If you use a clone from the GBU in your publication, please acknowledge us in the following way:

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

Please also cite the creators of the vector backbone of your construct. Details can be found on Addgene site for each plasmid:
lentiCRISPR v2