Integrated epigenetic and genetic programming of primary human T cells – Nature Biotechnology

TITLE: Epigenetic Engineering Breakthrough Enables Permanent Gene Silencing in Human Immune Cells

Revolutionary CRISPR Technology Transforms T Cell Programming

Scientists have achieved a major breakthrough in genetic engineering by developing a method for durable, specific silencing of endogenous genes in primary human T cells. Published in Nature Biotechnology, this research demonstrates how CRISPRoff technology can create stable epigenetic modifications that persist through multiple cell divisions, opening new possibilities for cell therapies and genetic research.

Optimizing CRISPRoff for Human T Cells

The research team systematically optimized multiple components of the CRISPRoff system for primary human T cells, testing seven different mRNA designs incorporating various cap modifications (Cap1, ARCA, and mg), base modifications (1-Me ps-UTP), and codon optimization algorithms. The CD151 cell surface protein served as the initial test case due to its well-characterized CpG island promoter and previous validation in HEK293T cells.

Critical optimization findings revealed: All base-modified mRNAs demonstrated superior CD151 knockdown compared to unoptimized versions. CRISPRoff 7 mRNA emerged as the most potent design, combining ‘design 1’ codon optimization, Cap1 mRNA cap, and 1-Me ps-UTP substitution. This configuration achieved complete silencing in 85-99% of cells without observable cellular toxicity, even at low mRNA concentrations., according to recent studies

Durable Silencing Through Multiple Cell Divisions

The researchers conducted extensive comparisons between CRISPRoff, CRISPRi, and Cas9 systems across multiple time points and nucleofector pulse codes. While CRISPRi produced only transient gene silencing that diminished over time, particularly after T cell restimulation, CRISPRoff programmed durable gene silencing comparable to permanent Cas9 knockout., as our earlier report, according to technological advances

Remarkable stability was observed: CRISPRoff maintained absence of cell surface expression in over 93% of cells for each targeted gene (CD151, CD55, and CD81) throughout the 28-day experiment, persisting through three separate restimulations with anti-CD2/CD3/CD28 soluble antibodies. This represents stable propagation of gene silencing memory across approximately 30-80 cell divisions in vitro., according to market analysis

Validating Specificity and Precision

The team employed multiple validation methods to confirm the specificity of CRISPRoff-mediated silencing. RNA sequencing analysis demonstrated highly specific repression, with only the targeted genes showing significant expression changes at 28 days post-electroporation. Whole-genome bisulfite sequencing further confirmed precise DNA methylation at target loci, with the highest differentially methylated region occurring specifically at transcription start sites.

When targeting therapeutically relevant genes including FAS, PTPN2, RC3H1, SUV39H1, MED12, and RASA2, the researchers found that multiple sgRNAs could potently and durably mediate CRISPRoff silencing. Analysis of potential off-target effects revealed exceptional specificity, with only one gene across 151 putative off-target sites showing evidence of potential off-target activity.

Expanding Beyond CpG Island Targets

Building on previous findings in HEK293T cells, the researchers explored whether CRISPRoff could silence genes lacking CpG islands in primary T cells. They targeted five non-CGI genes encoding important T cell surface proteins: CD5, LAG3, PDCD1, ENTPD1 (CD39), and PTPRC (CD45).

The results demonstrated varying but promising outcomes: For CD5 and LAG3, CRISPRoff silencing proved comparable to or even more efficient than Cas9 knockout, maintaining up to 99.5% and 99.1% silencing respectively at 30 days post-electroporation. PD1 silencing showed some variation between CD4 and CD8 T cells but remained stable in most cells across bulk populations.

Implications for Therapeutic Development

This research represents a significant advancement in genetic engineering capabilities for therapeutic applications. The ability to create stable, specific gene silencing without permanent DNA damage addresses key limitations of current CRISPR-Cas9 approaches. The technology enables reversible epigenetic modifications rather than irreversible genetic changes, potentially offering safer alternatives for cell therapies.

Key advantages identified include: No requirement for drug selection or cell sorting to identify modified cells, flexibility across multiple electroporation conditions, and compatibility with primary human T cells without compromising cell viability or proliferation. The system’s durability through multiple cell divisions makes it particularly valuable for therapeutic applications requiring long-term gene regulation.

The research team’s systematic optimization approach and comprehensive validation provide a robust framework for applying CRISPRoff technology to various therapeutic targets, potentially accelerating development of enhanced T cell therapies for cancer, autoimmune diseases, and other conditions.

For researchers interested in designing sgRNAs for similar applications, tools like IDT’s CRISPR design tool can help predict optimal targeting sequences and potential off-target effects.

References & Further Reading

This article draws from multiple authoritative sources. For more information, please consult:

• https://www.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE

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