Engineered PsCas9: a precise, efficient tool for therapeutic genome editing

Setting new benchmarks for safe and efficient therapeutic genome editing

What if curing genetic diseases became as straightforward as receiving a routine injection? It may sound like science fiction but breakthroughs in genome editing tools are bringing that future a little closer to reality.

The promise of CRISPR lies in its ability to precisely modify DNA, but concerns over efficiency and safety have limited its therapeutic applications. PsCas9, a high-fidelity Type II-B Cas enzyme, has demonstrated potential in genome editing with no detectable off‑targets and reduced chromosomal translocations, but exhibited limitations in efficiency when delivered using non-viral methods. This study by Degtev et al. (2024) showcases how engineering PsCas9 into engineered PsCas9 (ePsCas9) enhances editing performance while maintaining an exceptional safety profile. This innovation sets the stage for safer, more effective treatments for conditions like hypercholesterolemia and beyond.

Unlocking the power of ePsCas9: bridging efficiency and safety

Unlike the widely-used Streptococcus pyogenes Cas9 (SpCas9), PsCas9 boasts intrinsic precision, significantly reducing off-target effects. However, its efficiency lagged in non-viral delivery settings, a key limitation for clinical applications. Degtev et al. leveraged the high-resolution cryogenic electron microscopy (cryo-EM) structure of PsCas9's ribonucleoprotein, providing a basis for its rational engineering. They hypothesized that PsCas9’s modest editing activity could be attributed to its limited DNA binding ability. A combination of protein modification and single guide RNA (sgRNA) scaffold engineering boosted DNA binding and substantially improved PsCas9’s editing efficacy, leading to the creation of ePsCas9, with up to 20-fold enhanced activity.

With lipid nanoparticle (LNP) delivery, ePsCas9 demonstrated remarkable efficacy in editing the proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene in mouse liver and reducing plasma levels of Pcsk9 protein—a crucial step toward treating familial hypercholesterolemia.

Inside the engineered precision

The leap from PsCas9 to ePsCas9 involved two major advancements:

1. Protein modifications: strategic mutations achieved by introducing positively-charged amino acids to DNA interacting domains enhanced DNA binding, bolstering editing efficiency
2. sgRNA optimization: fine-tuned scaffolds reduced unnecessary structures, further improving target recognition

These adjustments preserved the enzyme's hallmark precision while minimizing off-target effects compared to SpCas9 and its high-fidelity variants, making ePsCas9 a safer alternative. Tests revealed a significantly lower risk of chromosomal translocations than SpCas9, setting a new standard for therapeutic genome editing.

But this is not just about an enzyme—it is an approach!

Creating ePsCas9 through rational engineering resulted in a high-fidelity and high-activity genome editor effective for in vivo applications using LNP delivery. But ePsCas9’s success is not just about one enzyme. This engineering strategy highlights the scalability of this approach for other Cas9 orthologs, expanding the CRISPR toolkit to develop efficient genome-editing therapies for a wider range of targets. From treating genetic disorders to advancing cell therapies, ePsCas9 offers a blueprint for safer, more effective genome-editing solutions.

As research progresses, tools like ePsCas9 could bring us closer to a future where genetic diseases are not just treated—but cured.

Reference

Degtev, D. et al. Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles. Nature Communications 15:9173 (2024)

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