CRISPRa cDNA or ORF

Which gene expression technology is best for my experiment?

Looking for the perfect complement to your loss-of-function studies? Maybe you’re gearing up for some biochemical studies? Need a tagged version of a gene for high-content imaging experiments?

There are many reasons you may want to overexpress your gene-of-interest. The traditional approach employed by molecular biologists is to exogenously express the gene of interest as a cDNA or ORF. The coding sequence is cloned into an expression vector under the control of a powerful promoter and delivered to the cells of interest by plasmid transfections or lentiviral particle transduction. Commercially available cDNA and ORF collections provide a quick and easily accessible method for obtaining gene content for many in vitro and cell culture applications. However, while this method is considered the gold standard, it does have its limitations. For example, the time it takes to clone and QC a gene if it is not already commercially available, or the risk of potential artifacts caused by non-native promoter effects.

The recent invention of CRISPR Activation (CRISPRa) has added a new overexpression tool to the toolbox. Utilizing a modified version of the S.pyogenes Cas9 protein (such as dCas9-VPR) in combination with a gene-specific guide RNA, CRISPRa can upregulate transcription of endogenous genes. This allows researchers to study genes in their native context--potentially leading to more biologically relevant results. However, this approach also has limitations, such as incompatibility for RNAi rescue experiments, and a requirement for accurate annotation of the gene-of-interest’s transcriptional start site(s).

How do you know which technology is right for you? Here is a brief list of the most common experiments involving overexpression, and recommendations for which method is preferred:

What are you trying to accomplish?

cDNA/ORF

CRISPRa

Express a tagged version of your gene in cells of interest

Yes

Maybe*

Want closest to natural post-transcriptional processing

No

Yes

Only want one isoform/variant expressed

Yes

No

Express a very long or difficult-to-clone gene

No

Yes

Need to express a non-protein coding gene (lncRNA, etc)

No

Yes

In vitro expression (protein purification, etc)

Yes

No

Rescue siRNA or shRNA knockdown

Yes

No

High-throughput overexpression screening

No

Yes

Human gene studies in animal models

Yes

No

Activate multiple genes in the same cell
*Cells of interest must already contain the tagged gene
** cDNA /ORF need to be cloned in vectors with different selection marker

Maybe**

Yes

If you are interested in learning more or discussing how cDNAs, ORFs or CRISPRa might be useful in your experiment, please just email or call our Tech Support team!

Authors: Jennifer Abarca is a Technical Support Scientist, Zaklina Strezoska is a Senior Scientist II at Dharmacon, Inc.

Additional Resources