Polymer-Stabilized Cas9 Nanoparticles and Modified Repair Templates Increase Genome Editing Efficiency

Purpose: CRISPR-Cas9 genome editing in clinically relevant primary cells is limited by inefficient homology-directed repair (HDR) and cytotoxicity of exogenous DNA. Improvements are needed to increase editing efficiency, cell viability, and generalizability across cell types for adoptive cell therapies and research.

Hypothesis: If truncated Cas9 target sequences (tCTSs) are added to the ends of HDR templates and Cas9 ribonucleoproteins (RNPs) are stabilized into nanoparticles with polyglutamic acid (PGA), then nuclear entry of the HDR template and editing efficiency will increase, toxicity will decrease, and lyophilized storage will be possible without loss of activity across multiple primary human hematopoietic cell types.

Aims: Develop a tCTS “shuttle” system to recruit Cas9-NLS RNPs to HDR templates and enhance nuclear entry. - Stabilize Cas9 RNPs into nanoparticles using anionic polymers, particularly polyglutamic acid (PGA). - Combine tCTS-modified HDR templates with PGA-stabilized RNPs to improve knock-in efficiency and viable edited cell yield. - Evaluate editing across multiple genomic loci and diverse primary human hematopoietic cell types. - Assess off-target editing and integration risks. - Test lyophilization and storage stability of PGA-stabilized RNPs.

Approach: Ex vivo human primary cells: Bulk CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer (NK) cells, and primary and iPS-derived CD34⁺ hematopoietic stem progenitor cells (HSPCs). - Genomic loci: TRAC, RAB11A, CD4, TUBA1B, ACTB, FBL, CLTA, IL2RA, IL2RG, and others. - Delivery: Electroporation of Cas9 RNP with or without PGA and with unmodified dsDNA or tCTS-modified HDR templates. - Doses: HDR template 0.75–3 µg; Cas9 RNP at 2:1 gRNA:Cas9 protein ratio unless noted. - Readouts: Flow cytometry for reporter expression and cell viability; amplicon sequencing for indels/off-target; DLS for particle size. - No in vivo animal model. No disease challenge model. - Sample size: n = 2–4 biologically independent human blood donors for most experiments.

Key methods: Flow cytometry: Knock-in efficiency, cell viability, edited cell recovery. - Amplicon sequencing: On-target and off-target indel analysis (CRISPResso2). - Dynamic light scattering (DLS): Nanoparticle size and colloidal stability. - Electroporation: Delivery of Cas9 RNP and HDR templates. - Lyophilization: Freeze-drying of PGA-stabilized RNPs; reconstitution and activity testing. - Statistical analysis: Mann–Whitney, two-way paired t-test with Holm–Sidak correction, two-way ANOVA; p < 0.05 considered significant.

Key results: tCTS shuttle: Facing orientation of tCTS on both 5′ and 3′ homology arms improved knock-in at TRAC. Representative flow cytometry: dsDNA vs tCTS — 45.7% vs 68.7%, 16.9% vs 54.1%, 18.7% vs 47.3%, 24.1% vs 51.3% across loci. - Across loci and T cell subsets: tCTS improved targeting across eight genomic loci in CD4⁺ and CD8⁺ T cells. - PGA stabilization: Anionic polymers including PGA enhanced editing; PGA-stabilized RNPs improved HDR and reduced toxicity. PGA-stabilized RNPs could be lyophilized and stored dry at −80°C with retained activity. - Combined system: Increased HDR and viable edited cell yields by ~2–6 fold. Bulk CD3⁺, CD4⁺, CD8⁺, and Tregs achieved >50% knock-in with 3–8 fold increase at reduced HDR template doses. NK and B cells: >15% transgene-positive, 2–5 fold increase. γδ T cells: ~5% to ~28%, 5–6 fold improvement. HSPCs: >15% large transgene insertion, 2–3 fold increase. - Off-target: PGA caused only slightly increased off-target indels at known EMX1 off-target sites. tCTS did not increase off-target transgene integration compared with standard dsDNA templates. - Lyophilization: PGA-stabilized Cas9 RNPs protected through lyophilization and retained activity.

Interpretation: The authors claim that combining PGA-stabilized Cas9 RNP nanoparticles with tCTS-modified HDR templates provides a technically simple, non-viral platform that greatly enhances genome targeting in primary human hematopoietic cells. The system increases editing efficiency and viable edited cell yield across multiple cell types and loci, with minimal increases in off-target events, and enables lyophilized storage. It has direct translational potential for research, biotechnology, and clinical applications, including next-generation adoptive cell therapies beyond T cells.

Limitations: No in vivo validation: All experiments were ex vivo in primary human cells; no animal models or in vivo editing. - Variable efficiency: Targeting success varied by locus, knock-in sequence, electroporation parameters, and cell type. - Some cell types remain challenging: B cells and NK cells achieved >15% but lower than T cells; further optimization may be needed. - Off-target assessment limited: Only selected off-target sites for EMX1 and functional integration tests were examined; global off-target edits/integrations for specific RNPs and templates need further work. - tCTS dose toxicity: tCTS-modified templates showed decreased viability at lower DNA doses, though PGA stabilization mitigated this. - Electroporation required: Delivery still depends on electroporation, which may limit scalability or certain cell types. - No clinical translation yet: Human primary cells only; no GMP manufacturing or clinical trial data. - Patent/competing interests: Several authors have patents, company affiliations, and competing interests.