@dlwagnerlabs.bsky.social on tour! 🌍🇩🇪🇺🇸

We had a great time at the Annual Congress of the European Society for Gene and Cell Therapy #ESGCT2025 in Sevilla. 🇪🇸

Super proud of everyone who presented their projects & grateful for working with this amazing team: science and good vibes! 🧬🧪💙

We found that the serine integrase Pa01 can be improved by adding a nucleus localization signal at the c-terminus -a small change that lifts Pa01 en par with the evolved Bxb1 variant from the Liu lab (eeBxb1). Engineered integrases boost 'one-pot' PASTA 🍝🛠️🚀

'one-pot' PASTA is more efficient than non-viral HDR and also better than PASSIGE with large constructs in primary human T cells. 🔬🚀

Super versatile; exchanging just nuclease + template enables you to put your cargo in a different place. 📍🗺️

'one-pot' PASSTA builds on 4 simple reagents that are easy to source & GMP compatible!

1. CRISPR-cas nuclease
2. HDR template (for landing pad insertion)
3. serine integrase mRNA
4. plasmid with cargo

-> a single electroporation in proliferating T cells =
✨ Efficient integration of XL-genes ✨

Further, in serial tumor challenges, Ana Nitulescu found a trend towards better expansion of CAR T cells generated by ssDNA with CTS over dsDNA - maybe there is a functional advantage of ssDNA? 🧐 (4/5)

Thanks to @jcornlab.bsky.social, we validated higher CAR transgene knock-in rates with CTS-modified ssDNA donors via long-read sequencing. Most integration events with unmodified ssDNA were partial (!) integration events! See Fig. 4 for more. Need to figure out why those happen... (3/5)

Recap: Adding Cas12a binding sites to linear single-stranded DNA donors increased knock-in rate with large transgenes. This replicates and validates previous work with Cas9 targets sites (CTS) by Shy et al. @marsonlab.bsky.social tinyurl.com/mr7v6ntv

... What's new since our preprint? (2/5)