We are very excited about the array of future applications for this technology, including diving deeper into the mechanisms of inflammation and accelerating the development of next-gen myeloid cell therapies. Check out the preprint here: www.biorxiv.org/content/10.6...

Finally, our screens highlighted TNFAIP3 as a major negative regulator of inflammatory polarization, so we disrupted this gene by KO or BE to polarize macrophages toward an inflammatory state, which could augment CAR-macrophage effector function and cancer cell killing.

Excitingly, we also use this technology to develop ‘SLICeVLP’, an adaption of our screening platform, SLICE, for LOF screens in primary human myeloid cells. Two pooled CRISPR screens illuminated regulators of cytokine production and inflammatory polarization in human macrophages.

We show high-efficiency base editing and epigenetic gene silencing applications too. Then, we combined VLP Cas9 delivery with AAV6 HDR donor template delivery to achieve site-specific transgene integration in primary human macrophages, which has been limited due to their post-mitotic nature.

Attempts at myeloid CRISPR editing have been hampered by cell loss & functional impairment. But, delivery of Cas9-RNPs using engineered virus-like particles (VLPs) achieved high-efficiency gene KO (to >99%), maintaining cell recovery and responsiveness to innate immune stimuli in human myeloid cells

Congratulations to Mineto Ota (now a PI in Tokyo) and whole team of scientists and many thanks to Jonathan Pritchard @jkpritch.bsky.social for being an inspiring partner.

Looking forward, we hope to apply this approach to understand genetic variants and programs regulating the immune system as we expand into genome-scale perturbations in human primary immune cells (stay tuned...)

Marrying perturb-seq with human genetic data allowed Mineto to link human genetic variants to molecular effects to human traits!

This establishes a new framework to gain biological insights into human genetic variants and gene programs that control key human traits in health and disease.

Since K562 cells are derived from a CML, they could be used to model features of human RBC differentiation. So he leveraged published genome-scale K562 perturb-seq (Jonathan Weissman) to interrogate gene programs controlled by genes with natural human mutations (UK biobank) that affect RBC traits.

For almost a decade Jonathan and I have been working together to gain insights into human genetics by building experimentally-informed gene regulatory networks. Mineto recognized that Perturb-seq allows us to test every gene in genome systematically and identify the gene programs each one controls.

We also show combinatorial genetic and epigenetic engineering with CRISPR Cas12a-dCas9 to perform targeted CAR knock-in with CRISPRoff-silencing of target genes to improve preclinical CAR T performance. Read the full paper here: rdcu.be/eL0GK

Using an all-RNA platform, we achieve efficient, durable, and multiplexed epigenetic programming in primary human T cells without the need for sustained expression of CRISPR machinery. The epigenetic changes are stable through cell divisions, cell stimulations, and after in vivo adoptive transfer.