Skip to main content

CRISPR HDR Breakthrough with mbDNA™: ssDNA Template Boosts Cell Viability & Scalability

By July 29, 2025Resources3 min read

CRISPR HDR Breakthrough with mbDNA™: ssDNA Template Boosts Cell Viability & Scalability

 

 

CRISPR has transformed our ability to edit genomes, enabling targeted insertions, deletions, and base corrections. Yet implementing homology-directed repair (HDR) in therapeutic settings remains fraught with technical and logistical hurdles. From donor-to-donor variability to immunogenic responses, existing DNA templates often struggle to balance editing efficiency, cell viability, and manufacturing scalability.   

The CRISPR HDR Bottleneck in Cell Therapy

Delivering reliable, high-efficiency HDR in immune or stem cells typically encounters:   

  • Donor-to-donor variability in cell viability, recovery, and yield   
  • Suboptimal knockout/knock-in (KO/KI) rates that lengthen development cycles   
  • Immunogenic sequences triggering unwanted immune activation   
  • Inefficient nuclear import of non-viral DNA   
  • Size constraints limiting cargo size to a few kilobases   

Overcoming these intertwined challenges demands a next-generation template designed from the ground up for precision and performance.   

Introducing mbDNA: A Tailored ssDNA HDR Platform

mbDNA (Megabulb DNA) rethinks CRISPR HDR templates as circular, single-stranded DNA molecules with a brief, user-defined double-stranded stem (~30 bp).

This architecture:

  • Supports large gene-of-interest (GOI) cargos up to 20 kb   
  • Includes a short-hairpin structure for robust Cas nuclease binding   
  • Promotes active nuclear import for efficient template delivery   

By combining precise gene insertion with a minimal immunogenic footprint, mbDNA enables consistent cell viability and high KI rates across diverse donors.   

Scalable, Cell-Free Manufacturing

Traditional ssDNA synthesis at gene length is laborious and low yielding. Touchlight’s proprietary cell-free DNA platform addresses this by:   

  • Utilising enzymatic amplification for rapid, high-fidelity ssDNA production   
  • Offering full control over sequence composition and batch consistency   
  • Accelerating timelines for clinical applications with GMP coming soon

This streamlined process bridges research and manufacturing, ensuring that HDR templates are available at scale without compromising quality.   

Proven Performance in the Generation of CAR-T Cells

In collaboration with an autologous ex vivo CAR-T cell partner, GMP-representative mbDNA was benchmarked against dbDNA™ and plasmid DNA (pDNA). Key findings included:   

  • Knock-in efficiency reaching up 70% across a broad concentration range (see Figure 1)    
  • Cell counts on par with DNA-free controls, indicating minimal cytotoxicity (see Figure 2) 
  • Robust GOI integration at the TRAC locus, measured eight days post-electroporation  
Percentage of knock-in positive cells of mbDNA was consistently higher than pDNA controls across different donors. Asterisks indicate conditions where cell viability was too low to yield usable data. 
Figure 1: Percentage of knock-in positive cells of mbDNA was consistently higher than pDNA controls across different donors. Asterisks indicate conditions where cell viability was too low to yield usable data. 
Following intracellular delivery, mbDNA consistently allows for high cell survival, recovery, and expansion in comparison to pDNA.
Figure 2: Following intracellular delivery, mbDNA consistently allows for high cell survival, recovery, and expansion in comparison to pDNA. 

Shaping the Future of Gene Therapy

mbDNA sets a new standard for non-viral HDR templates by uniting molecular innovation with scalable manufacturing. Its ability to deliver precise editing, preserve cell health, and support larger cargo paves the way for more effective, accessible cell and gene therapies.   

With GMP coming soon, leading biopharma and biotech organisations are already exploring mbDNA across preclinical platforms. By integrating this technology into your workflow, you can accelerate development, minimise risk, and move toward clinical impact with confidence.   

To learn how mbDNA can transform your gene-editing programs, read our new mbDNA eBook or contact our team.