A Biological Hijack

October 2, 2024 by Patrick Paez

Activating DNA repair mechanisms with CRISPR

In our latest whitepaper, Next-generation CRISPR Approaches, we discuss the various Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) approaches and how some newer concepts are changing how we approach the modality. We start with the basic mechanism of how Cas nucleases bound to a guide RNA (gRNA) form and ribonucleoprotein (RNP) are capable of generating a targeted double-stranded break in the DNA in a cell.

This results in the activation of DNA repair mechanism which will dictate a knock-out of a gene or the knock-in of a desired gene of interest when supplying the cell with a template for repair.

Expanding on that thought, we discuss in detail the hijacking of biology to knock-out or knock-in genes through:

• Homology directed repair
• Homology independent targeted insertion
• Microhomology end joining

These knock-in pathways are being explored using various vector systems to deliver the RNP and donor template, providing various avenues to edit target cells in all phases of the cell cycle.

Which vector should I choose for my CRISPR system?
Selecting an approach for RNP and donor template delivery is highly dependent on the target cell type and the setting of the editing (i.e. ex vivo or in vivo). We discuss the benefits and potential limitations inherent in deploying several common approaches including viral vector and non-viral vector delivery.

We also look at new and novel approaches using next-generation DNA vectors like Nanoplasmid™, used in generating knock-in rates between 70-90% in the ex vivo setting.

Considerations for selecting a CDMO partner for your CRISPR therapy
When deciding on a CRISPR approach, whether to go viral or non-viral or a combination of the two is just part of the difficult decisions developers must contend with.

Partnering with the right CDMO that can offer next-generation technologies to improve your workflow, off-the-shelf nucleases with consistent biological activity across quality grades, and a first-in-class RNP manufacturing and analytical service to meet FDA requirements, is option that can improve the chances of clinical success of your CRISPR-based therapy.

Download Next-generation CRISPR Approaches for a discussion of the most common CRISPR approaches and novel knock-in methods designed to achieve high knock-in rates.

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• Visit our Nanoplasmid page
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