Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency

Area of Science:

• Biotechnology
• Molecular Biology
• Immunotherapy

Background:

• Precise genome editing is crucial for developing advanced adoptive cellular therapies.
• Current CRISPR-Cas9 methods face efficiency limitations in clinically relevant primary cell types.

Purpose of the Study:

• To enhance the efficiency and applicability of CRISPR-Cas9 genome editing in primary cells.
• To develop improved strategies for homology-directed repair (HDR) in cellular therapies.

Main Methods:

• Introduction of truncated Cas9 target sequences (tCTSs) into HDR templates to facilitate nuclear import via Cas9 ribonucleoproteins (RNPs).
• Stabilization of Cas9 RNPs into nanoparticles with polyglutamic acid to improve editing and reduce toxicity.
• Application of combined enhancements across diverse primary cell types, including T cells, B cells, NK cells, and hematopoietic stem progenitor cells (HSPCs).

Main Results:

• HDR efficiency was enhanced two- to fourfold by tCTS-mediated template shuttling.
• Nanoparticle-stabilized Cas9 RNPs further improved editing efficiency twofold, reduced toxicity, and allowed for lyophilized storage.
• The combined approach yielded two to six times more viable edited cells across multiple genomic loci and cell types.

Conclusions:

• The developed CRISPR-Cas9 editing enhancements significantly improve gene targeting efficiency in clinically relevant primary cells.
• These improvements offer a more robust and versatile platform for generating cells for adoptive cellular therapies.
• The methods are applicable to a wide range of immune and stem cells, broadening therapeutic potential.