Feng Zhang Profile picture
Mar 29, 2023 8 tweets 4 min read Read on X
Delivery of therapeutic molecules is a major bottleneck for treating a wide range of diseases. Today we describe a new modality for delivering proteins based on an engineered contractile injection system @nature nature.com/articles/s4158…
1/ Inspired by the machinery used by endosymbiotic bacteria to deliver protein payloads to their host animals’ cells, we sought to engineer these systems, called contractile injection systems (CISs), for delivery to human cells.
2/ We focused on the Photorhabdus virulence cassette (PVC), a type of CIS that is secreted by Photorhabdus and known to infect insect and mouse cells. We reconstituted PVCs in E. coli and confirmed they could attach to insect cells, their native target.
3/ We then explored if we could replace the endogenous cargo protein with a protein of interest. To do this, we introduced a packaging signal on a cargo protein (GFP, Cre, or ZFN), confirmed it was loaded by the PVC, and showed it functioned in recipient insect cells.
4/ To redirect the tropism of PVCs to human cells, we took advantage of the structural similarities between the PVC and contractile phage tails, modification of which can change phage targeting. Based on this, we focused our engineering efforts on Pvc13, the tail fiber protein.
5/ Using #AlphaFold to model Pvc13, we predicted which part of Pvc13 contacts target cells and used this information as a guide to insert binding domains specific to human cells into Pvc13. We confirmed these PVC variants bound to and delivered cargo to the intended target cells.
6/ We took a similar structure-guided approach to target neurons in the mouse brain, showing in vivo delivery of a cargo protein specifically to neurons. We are continuing to expand the targeting and packaging abilities of PVCs and test them in vivo.
7/ This work was led by graduate student Joe Kreitz (@kreitz_joseph) with collaboration from MFriedrich (@mircoscopy), AGuru (@Akash_Guru_), BLash (@blake_lash), and MSaito.

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More from @zhangf

Jun 28, 2023
Since the discovery of CRISPR-Cas systems, people have wondered if a similar type of system exists in eukaryotes. Today, we report in @Nature the characterization of Fanzor, a eukaryotic RNA-guided system that can be reprogrammed for human genome editing. nature.com/articles/s4158…
1/x In exploring the evolution of Cas9, we identified Fanzor (Fz) as a putative RNA-guided nuclease while studying IscB, IsrB, and TnpB–collectively called OMEGA systems–in collaboration with Eugene Koonin’s group. Here, we expand our analysis of the diversity of Fz proteins,… https://t.co/7XIcScOPlhtwitter.com/i/web/status/1…
2/x Fz1 and Fz2 share similar domain architecture and catalytic residues with the OMEGA effector TnpB and the CRISPR effector Cas12, and our analysis suggests TnpB evolved into both prokaryotic Cas12 and eukaryotic Fz.
Read 10 tweets
Sep 9, 2021
CRISPR technologies are powerful gene editing tools because they are programmable. We are excited to describe 3 new programmable systems, IscB, IsrB, and TnpB, some of the most abundant genes on the planet, with potential for gene editing. @ScienceMagazine science.org/doi/10.1126/sc…
1/ We were exploring Cas9 diversity when we found IscB, a transposon-encoded protein that shares nuclease domains with Cas9. However, unlike Cas9, no RNA or CRISPR components are known to interact with it, making its function a mystery.
2/ Using both computational and experimental approaches, we found IscB associates with a new, diverse class of large structured non-coding RNAs, termed omegaRNAs.
Read 9 tweets
Aug 19, 2021
We are really excited to share our work on a new modular RNA delivery technology called SEND, built from endogenous components found in the mammalian genome. @ScienceMagazine science.sciencemag.org/content/373/65… Image
1/ Delivery of nucleic acid therapies to cells remains a bottleneck for gene therapy. We wanted to develop a modular delivery system that would be safe and cell-type type specific, avoiding the immunogenicity of common viral vectors. Image
2/ Given cool findings by Vivian Budnik/Travis Thomson and @JasonSynaptic showing the retrotransposon-derived protein ARC forms capsids and transfers mRNA between cells, we collaborated with Koonin lab to identify other retroelement-derived proteins that might form capsids. Image
Read 9 tweets
May 5, 2020
We are excited to share STOPCovid, an inexpensive, one-step, & sensitive #COVID-19 detection protocol developed with @omarabudayyeh & @jgooten. This revamped assay was streamlined with the future goal of developing an in-home test for COVID-19. zlab.bio/s/COVID-19-det… 1/X
#STOPCovid requires no sample extraction, can be performed using a single mastermix, and uses a paper strip to display results. #STOPCovid can detect down to 100 copies of SARS-Cov-2 RNA from patient samples. 2/X
Enabling testing at home and in low-resource settings requires that we can’t be reliant on complex instrumentation. #STOPCovid can be used with a sous-vide cooker (<$40). 3/X
Read 7 tweets

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