Adenoviral Delivery of CRISPR/Cas9 Aims to Expand Genome Editing to Primary Cells

Posted by Kendall Morgan on Sep 30, 2014, 4:50:49 PM

Adenoviral-CRISPR-Cas9-delivery-plasmids-Manuel-GoncalvesResearchers have shown that it is possible to deliver RNA-guided CRISPR/Cas9 nuclease complexes using adenoviral vectors (AdVs), to a wide range of human cells, including mesenchymal stem cells, and in a rather straightforward manner. These adenoviral CRISPR/Cas9 genome editing tools developed and demonstrated by Manuel Gonçalves and his colleagues at Leiden University Medical Center are now available at Addgene along with a description of their experimental protocol. The three plasmids which have been deposited to Addgene are: pAdSh.PGK.Cas9, pAdSh.U6.gRNAS1, pAdSh.U6.gRNAGFP.

“Although AdVs are being deployed for delivering zinc-finger nucleases into human cells, we think they are still underused in the emerging field of genome editing,” Gonçalves said. “In contrast, AdVs are extensively being explored for genetic vaccination and oncolytic approaches. In genome editing, they are not used much, but we do think they have a very bright future.”

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Gonçalves says that advantages of AdVs include their episomal nature and very efficient introduction of DNA into therapeutically relevant, non-transformed mammalian cells. These viral vector systems also work equally well in dividing and quiescent, post-mitotic mammallian cells.

Delivering gRNA and Cas9 using adenoviral vectors

In a Scientific Reports paper introducing the delivery method in May, the researchers report that AdV-mediated transduction of gRNA:Cas9 ribonucleoprotein complexes into transformed and non-transformed cells yielded rates of targeted mutagenesis similar to those achieved by isogenic AdVs encoding TALENs targeting the same chromosomal region. The CRISPR/Cas9-derived RNA-guided nuclease-induced gene disruption frequencies in the various cell types ranged from 18% to 65%.

A second paper published online in Nature Methods in August found that delivering RNA-guided nucleases or TALENs together with AdV donor DNA leads to a vast majority of AdV-modified human cells being subjected to scarless homology-directed genome editing. Gonçalves said they attribute this phenomenon to the presence of terminal proteins capping the ends of linear double-stranded AdV genomes. Such protein-DNA structures presumably reduce the likelihood that donor DNA will interact with sporadic double-stranded chromosomal DNA breaks “that always happen naturally.”

“We think this [most recent] work gives additional rationale for investigating the usefulness of adenoviral vector technology in the context of genome editing,” he said, adding that he hopes others will now begin to make use of the new AdV delivery tools for a variety of applications.

“It would be rewarding if these reagents and protocol are picked up and people start to explore and test this method of introducing the CRISPR system into a broader range of cells – primary cells and cells that are not transformed – and eventually also consider in vivo applications.”

Start using adenoviral vectors with your CRISPR/Cas9 research!

To find more information about the adenoviral delivery of CRISPRS/Cas9 using the Gonçalves lab's plasmids, including protocols, check out the plasmids at Addgene: pAdSh.PGK.Cas9 (expresses S. pyogenes Cas9 from the PGK promoter) and U6 promoter-driven guide RNA constructs, pAdSh.U6.gRNAS1 and pAdSh.U6.gRNAGFPOr if you're looking for a broader range of CRISPRs plasmid tools, find more plasmids, CRISPR technology guides, FAQs, and CRISPR resources on Addgene's site:



1. Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells. Maggio I, Holkers M, Liu J, Janssen JM, Chen X, Gonçalves MA.  Sci Rep. 2014 May 29;4:5105. doi: 10.1038/srep05105. PubMed PMID: 24870050. PubMed Central PMCID: PMC4037712.

2. Adenoviral vector DNA for accurate genome editing with engineered nucleases. Holkers M, Maggio I, Henriques SF, Janssen JM, Cathomen T, Gonçalves MA. Nat Methods. 2014 Oct;11(10):1051-1057. doi: 10.1038/nmeth.3075. Epub 2014 Aug 24. PubMed PMID: 25152084.

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