Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome...

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Gene editing for medicine faces challenges from enzymes and immune responses.
• Previous work identified novel gene-editing systems from metagenomic data.
• Further development is needed to overcome limitations in current gene-editing technologies.

Purpose of the Study:

• To demonstrate the utility of three novel gene-editing systems for cell therapy development.
• To evaluate the efficiency, specificity, and safety of these systems in various primary immune cells.
• To assess the potential for integrating therapeutic constructs like chimeric antigen receptors (CARs).

Main Methods:

• Utilized three novel gene-editing systems for high-frequency gene editing in human T cells, NK cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells.
• Disrupted target genes including TCR alpha-chain, TCR beta-chain, B2M, TIGIT, FAS, PDCD1, TRAC, and TRBC.
• Integrated a CAR construct into TRAC and assessed CAR expression and cytotoxicity.
• Evaluated gene-editing specificity and compared it to Cas9.
• Assessed for pre-existing humoral and T cell-based immunity against the nucleases.

Main Results:

• Achieved reproducible, high-frequency gene editing (>90-95%) in primary human immune cells, including simultaneous double knockouts.
• Demonstrated minimal impact on T cell viability post-editing.
• Successfully generated CAR-expressing T cells and CAR-NK cells with demonstrated cytotoxicity.
• Observed gene-editing specificity comparable to or better than Cas9.
• Confirmed lack of pre-existing immunity against the novel nucleases.

Conclusions:

• The novel gene-editing systems exhibit high activity, specificity, and translatability for cell therapy.
• These systems overcome previous enzymological and immunological impediments in gene editing.
• The demonstrated efficiency and safety profile support their application in developing next-generation cell therapies.