Advancing the safety of CRISPR as a potential future therapeutic

Written by:

Ben Taylor

Associate Director, Discovery Sciences, BioPharmaceuticals R&D


Adding to our growing arsenal of tools with the aim of bringing the promise of CRISPR-based therapeutics to the clinic, our latest publication in Nature Communications introduces CRISPR GUARD, a novel tool that, using short inactive guide RNAs, has demonstrated the potential to nearly eliminate off-target editing in a pre clinical setting. This approach has the potential to improve the specificity and safety of CRISPR for possible future therapeutic use.


Preclinical investigations to support potential applications of CRISPR in future

In CRISPR, molecular complexes called Cas9 and base editors are used to make edits in DNA and can be programmed using guide RNAs to precisely direct them within the genome. Imagine molecular scissors, for example, cutting and correcting misprints (mutations) in our genetic code. Precise genome editing using CRISPR is emerging as a potential therapeutic possibility for cell therapies and genetic diseases.

While gene editing with CRISPR-Cas9 is simpler to execute and much more precise compared with other DNA-modifying tools, the chief concern is mis-direction of these molecular scissors to similar but incorrect sites (off-targets).

Inspired by the discovery that guide RNAs shorter than 16 nucleotides can direct Cas9 but do not allow gene editing activity – effectively shielding the DNA from cutting, we looked at directing these short guide RNAs (or GUARD RNAs) to known off-target sites to protect them from unwanted editing. We call this method CRISPR Guide RNA Assisted Reduction of Damage, or CRISPR GUARD.

In a new study, published in Nature Communications, we tested the effectiveness of hundreds of GUARD RNAs in cell models.1 Encouragingly, we showed that most off-targets can be protected by carefully designed GUARD RNAs, without any impact on on-target editing at the desired site. Our results have also been confirmed independently by other labs, helping establish the universality of this approach.2

Conventional methods to avoid off-target cutting rely on modifying Cas9 enzymes to create high-fidelity variants that can no longer cut at off-target sites. Whilst this works very well to minimise the risk of off-target editing, it doesn’t fully eliminate the possibility of undesired editing at every potential off-target site. Early data from our research and others indicates that CRISPR GUARD can be combined with these high-fidelity Cas9 variants to nearly eliminate off-target editing.



Safety-first CRISPR toolbox

These new data build upon previous work from our scientists and collaborators that have helped us understand the impact of off-target editing using CRISPR.

In 2019, ‘DISCOVER-Seq’ was published in Science; this tool allows us to directly monitor CRISPR-based genome editing in human cell- and mouse-models.3 By tracking sites of DNA breaks, it is able to highlight off-target sites with high specificity. In our most successful experiment at the time, we accurately identified 36 off-target events, compared with a list of over 3,000 sites with false positive outcomes detected with previous methods.

Prior to that, we described a novel approach for ensuring the specificity of gene editing, which we called CRISPR VIVO (Verification of In Vivo Off-targets).4 We designed guide RNA sequences to be highly selective to the target gene sequence and were able to show, in relevant preclinical models, no detectable off-target modifications throughout the genome, thus demonstrating the importance of using well-designed high-fidelity guide RNAs in CRISPR.

Taken together, this underscores our commitment to CRISPR safety. With CRISPR GUARD in particular, we foresee potential applications in cell therapies or functional gene editing in future, to protect off-target sites that might not be avoided by other mechanisms. With the hope of making CRISPR GUARD a widely applied tool in gene editing research, we have also developed an online tool to design GUARD RNAs for a given off-target sequence: https://www.sanger.ac.uk/science/tools/crisprguardfinder/crisprguardfinder/


References

1. Coelho, M.A., De Braekeleer, E., Firth, M. et al. CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs. Nat Commun 11, 4132 (2020). https://doi.org/10.1038/s41467-020-17952-5.

2. Rose, J.C., Popp, N.A., Richardson, C.D. et al. Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs. Nat Commun 11, 2697 (2020). https://doi.org/10.1038/s41467-020-16542-9.

3. Wienert, B. et al. Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. Science, doi:10.1126/science.aav9023 (2019).

4. Akcakaya, P. et al. In vivo CRISPR editing with no detectable genome-wide off-target mutations. Nature 561, 416-419, doi:10.1038/s41586-018-0500-9 (2018).


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