Production of virus-like particles with AsCas12a nuclease and CMV-driven crRNA for mammalian genome editing

Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells.

A virus-like particle (VLP)-based toolkit that delivers diverse CRISPR editing modalities to human monocytes, macrophages and dendritic cells with high efficiency while preserving viability and innate immune responsiveness is presented.

Co-delivery of lentiviral vectors and Cas9-containing virus-like particles enables rapid, scalable manufacture of gene-edited CAR T cells

Chimeric antigen receptor T cell immunotherapies are transforming therapies for hematological malignancies and solid tumors and can be enhanced by targeted gene knockout. Here, we report lentiviral-based virus-like particles that package and deliver Cas9 ribonucleoproteins to primary human T cells. Using distinct pseudotyping strategies for virus-like particles and for lentiviral or γ-retroviral vectors, we achieved chimeric antigen receptor expression and targeted gene disruption. Under optimized transduction conditions, more than 50% of T cells expressed a chimeric antigen receptor by flow cytometry, with vector copy numbers exceeding two. Editing efficiencies were above 70% at three different target loci tested: T cell receptor α constant chain, β2-microglobulin, and DNA methyltransferase 3α. When the editing efficiency of virus-like particles was directly compared to electroporation, electroporation achieved a higher editing efficiency (99% versus 70%–90%). However, virus-like particle treatment resulted in twice as many cells being recovered compared with electroporation with a 10% increase in cell viability. Furthermore, off-target editing in virus-like particle-treated cells was reduced compared to ribonucleoprotein electroporated cells. These results support the feasibility of using virus-like particle-mediated delivery of Cas9 ribonucleoprotein to disrupt genes of interest, enabling a more scalable and cost-effective process for generating T cell immunotherapies.

Virus-like particle-delivered base editor collection to expand the genome engineering toolbox

Virus-like particles (VLPs) enable transient, non-integrating delivery of CRISPR-Cas9 ribonucleoprotein cargo. Although VLPs have been reported for efficient DNA editing via base editors RNP delivery, the diversity of base editors tested as VLPs remains limited. We generated and benchmarked a panel of 12 base editors on the v5 eVLP backbone, targeting three genomic loci (HEK3, B2M, PDCD1) across five VLP dosages in LentiX-293T cells. Editing efficiency was generally dosage-dependent across all editors and varied by editor class and identity; PAM-flexible variants had lower editing efficiency than NGG-restricted counterparts, and the dual-function SPACE base editors showed reduced efficiency. We further characterized position-specific editing efficiencies and outcomes of the base editor VLP collection, revealing that a wide variety of mutation types are possible with the base editors in this collection.

Establishment of a scalable engineered cell-line platform for direct, GMP-grade production of eVLP vectors enabling streamlined generation of gene-edited CAR-T/NK cells

Introduction CRISPR–Cas9 has transformed the engineering of chimeric antigen receptor T (CAR-T) cells and chimeric antigen receptor NK (CAR-NK) cells; however, its clinical translation remains constrained by the high cost, batch-to-batch variability, and stringent regulatory requirements associated with current viral and electroporation-based manufacturing approaches. Methods We report an industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9–gRNA ribonucleoproteins (RNPs). A progenitor cell line was established by stably integrating three core modules—Gag-Pol, Gag-Cas9, and the baboon endogenous virus (BaEV) envelope—into a single HEK293T clone. Introduction of a self-inactivating (SIN) retroviral vector encoding the gRNA cassette (exemplified here by CD7) converted this progenitor into a dedicated eVLP producer within 10 days. Results Using this platform, we generated CD7-knockout CAR-T/NK cells that retained robust in vitro cytotoxicity, confirming preserved functional activity. Owing to its modular architecture, the platform is readily extensible. For example, integration with Recombinant Adeno-associated Virus (rAAV) donor templates enables site-specific CAR insertion, while multiplexed eVLP cocktails allow simultaneous disruption of multiple genomic loci. Discussion It is worth noting that this workflow eliminates the need for electroporation, reduces serum dependency, and significantly lowers the cost of reagent consumables. Collectively, this system provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.

Efficient in vivo cytosine base editing using virus-like particles with uracil DNA glycosylase inhibition.

Compact 9dBEs Enable Efficient and Precise Genome Editing in Mammalian Cells and In Vivo

ABSTRACT Despite the promise of DNA base editors for diverse genome editing applications, their utility remains constrained by off‐target effects, which are exacerbated by short spacers in miniature systems and the large size of Cas9‐derived editors, which impedes adeno‐associated virus (AAV) delivery. Here, guided by structural insights into a compact Cas9d nuclease from Deltaproteobacteria, we developed an efficient Cas9d system (Cas9dUltra) through gRNA and protein engineering, and further developed its base editors (9dBEs). Cas9dUltra and 9dBEs enabled efficient and precise genome editing in human cells. Notably, 9dCBE induced premature termination codons in 89% of mouse pups by microinjection, facilitating robust disease modeling. Furthermore, a single AAV delivering 9dCBE achieved efficient Pcsk9 editing and a concomitant reduction in serum LDL‐C levels in mice. Collectively, this study establishes a series of compact, potent genome editing tools poised to advance biological and biomedical translational research.