We hope this will be a foundational resource for the HIV research community and also serve as a prototype for understanding infectious diseases in other human cell types.

Our lab believes there is no substitute for studying HIV in real, primary human CD4+ T cells. This study performs systematic discovery and mechanistic studies in these primary cells to uncover new host-pathogen biology.

And it's a testament to collaboration with Nevan Krogan, Harmit Malik and Vinay Pathak and the whole NIH HARC Consortium.

My lab has been studying HIV for over a decade, inspired by training as an infectious disease doctor who used to have an HIV clinic in SF. This work was led by Ujjwal Rathore and Eli Dugan, a truly incredible scientific duo.

Evolutionary data indicated that PPID from various non-human primates is even more effective as an HIV antiviral factor than human protein. We identified targeted mutation, including amino acid variants from in non-human primates, that can enhance PPID restriction factor activity.

PPID overexpression reduced nuclear import of HIV capsid, and in-depth domain mapping of PPID identified the protein domains and key amino acid residues responsible for HIV restriction by this protein.

But, PPID has not been studied in the context of HIV and we now reveal how its TPR domain, not found in CypA, is critical for its potent antiviral activity. Amazingly, fusion of the PPID TPR domain to CypA (which binds HIV capsid and promotes nuclear entry) converts CypA into anti-viral factor!

This is interesting from a virology perspective because PPIA (also known as CypA) is a related protein that is well-studied and critical for mediating proper HIV capsid entry into the nucleus.

We then did deep characterization of PPID, a novel antiviral factor. We found that PPID potently restricts both CXCR4- and CCR5-tropic HIV as well as VSV-G pseudotyped virus.

The result is a remarkably comprehensive map of human genes that serve as host factors for HIV infection, including anti-viral factors that had not been identified until now.

We systematically studied every gene to identify ones that can increase or decrease HIV infection in primary human CD4+ T cells. Importantly, these studies use CRISPR to disrupt each gene and then separately use CRISPR activation to increase the levels of each gene.

My lab has been studying human genes that control infection HIV infection of CD4+ T cells for over a decade. Today we report a major milestone in Cell: www.cell.com/cell/fulltex...

Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells Primary human myeloid cells are promising candidates for immunotherapy, yet efficient and scalable technologies for genetic engineering and screening in these cells are limited. Here we present a viru...

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

Good news keeps coming! Excited to share our latest preprint expanding myeloid cell editing capabilities: Virus-like particles enable targeted gene engineering and pooled CRISPR screening in primary human myeloid cells, led by Hyuncheol Jung and Pascal Devant joint with @juliacarnevale.bsky.social

Causal modelling of gene effects from regulators to programs to traits - Nature Approaches combining genetic association and Perturb-seq data that link genetic variants to functional programs to traits are described.

GWAS has been an incredible discovery tool for human genetics: it regularly identifies *causal* links from 1000s of SNPs to any given trait. But mechanistic interpretation is usually difficult.

Our latest work on causal models for this is out yesterday:
www.nature.com/articles/s41...
A short🧵:

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.

Our latest collaboration with @jkpritch.bsky.social – led by joint post-doc Mineto Ota – is in @nature.com today: www.nature.com/articles/s41...

Integrated epigenetic and genetic programming of primary human T cells Nature Biotechnology - Multiplexed editing in primary human T cells generates enhanced immune cell therapies.

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

Integrated epigenetic and genetic programming of primary human T cells Nature Biotechnology - Multiplexed editing in primary human T cells generates enhanced immune cell therapies.

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.

Integrated epigenetic and genetic programming of primary human T cells Nature Biotechnology - Multiplexed editing in primary human T cells generates enhanced immune cell therapies.

Happy to share our latest paper in Nature Biotech, led by graduate student Laine Goudy, joint with Luke Gilbert at Arc, developing CRISPRon/off technology for epigenetic programming of human T cells.