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David R. Liu

18 Feb, 10 tweets, 4 min read

@ScienceMagazine

Today we report in @ScienceMagazine the development of a continuous evolution system that can reprogram a family of clinically used proteases to selectively cleave protein targets of our choosing. 1/10
science.sciencemag.org/content/371/65…

drive.google.com/file/d/1PqaQSt…

Proteases offer powerful features as therapeutics, including potency as catalysts & the ability to modulate protein function, localization, & lifetime. But the difficulty of generating proteases that cleave proteins of our choosing has been a key barrier to their broader use.2/10

Phage-assisted continuous evolution (PACE) can rapidly evolve proteins with tailor-made activities or specificities over thousands of generations of evolution at the speed of dozens or hundreds of generations per day, with minimal required human intervention. 3/10

We developed a PACE system for protease evolution that includes both a positive and negative selection, allowing the system to rapidly evolve proteases that cleave a specified target protein and that do not cleave off-target protein(s). 4/10

We applied this protease reprogramming platform to evolve four proteases from three families of Botulinum neurotoxin light-chain proteases (BoNT proteases). BoNTs are already used as FDA-approved drugs to treat a variety of conditions. 5/10

The most challenging of these 4 campaigns was the evolution of a BoNT/E protease that cleaves PTEN, a protein of biomedical interest that is unrelated to any known substrate of any BoNT protease. This campaign took thousands of generations of positive & negative selection. 6/10

All four evolved proteases cleave their new substrates with high specificity over their original substrates, achieving changes in substrate specificity ranging from 218- to >11,000,000-fold, despite having no detectable starting activity on their new targets in two cases. 7/10

We also showed that BoNT proteases evolved in this system can retain their activity in mammalian cells and can retain their ability to combine with BoNT heavy chains to form BoNT holotoxins that potently self-deliver into primary neurons at picomolar concentrations. 8/10

This work may provide a foundation for the rapid generation of new proteases that each selectively cleave a target of therapeutic interest. Such a capability could be a step towards "editing the proteome" to complement technologies to edit the genome.(9/10)drive.google.com/file/d/1uPJYUm…

@trblum

Congratulations to Travis Blum (@trblum), Hao Liu, Michael Packer, Ziaozhe Xiong, Pyung-Gang Lee, Sicai Zhang (@sicaizhang), Michelle Richter, George Minasov, Karla Satchell (@microProfMom), and Prof. Min Dong for completing this first chapter of our collaboration! (10/10)

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David R. Liu

@davidrliu

6 Jan

Today we report the use of base editing in patient-derived cells and in mice to correct the most common cause of progeria, the devastating rapid aging disease. Progeria is typically caused by a dominant negative C•G-to-T•A point mutation in LMNA. 1/11 drive.google.com/file/d/1oH4W0z…

@NIHDirector

The mutation (discovered by @NIHDirector’s lab in 2003) results in progerin, a toxic protein that damages nuclei. So providing more healthy LMNA is not a solution, and cutting the mutated gene with nucleases is also challenging due to indel mixtures & similarity with wt LMNA.2/11

We used an adenine base editor to convert the T•A that causes progeria back to C•G at the key position in LMNA. Base editing in cells from progeria children corrected the mutation efficiently (~90%) with minimal indels or off-target edits, and restored nuclear morphology. 3/11

Read 11 tweets

David R. Liu

@davidrliu

27 Aug 20

@CDCgov

Bizarrely, @CDCgov now says Americans don’t need to get tested even if they have been in close contact with a #COVID19 infection, and that asymptomatic people do not need a test. More Americans will needlessly die as a result. Here's a thread on why testing is essential. (1/12)

@youyanggu

From one of the most accurate #COVID19 models to date (@youyanggu's covid19-projections.com/#current-us-pr…), about 1 in 75 Americans today has an active #COVID19 infection. Many of these are asymptomatic and unaware they are infected, but can still infect others. (2/12) nejm.org/doi/full/10.10…

Without identifying the 1/75 who has an active infection and separating them from the 74/75 who do not, we either allow the 1/75 to mix with others, causing new outbreaks and adding to 180,000 US deaths, or we lockdown, even though 74/75 lack the virus. Neither is acceptable.3/12

Read 12 tweets

David R. Liu

@davidrliu

24 Aug 20

https://twitter.com/cwylilian/status/1297857565385093121

A new report by Hong Kong University documents an apparent case of #COVID19 reinfection by two different #SARSCoV2 strains. A 33-year-old male presented symptoms and tested positive by RT-PCR on Mar 26. Hospitalized Mar 29, discharged Apr 14 after two negative PCR tests. (1/6)

https://twitter.com/cwylilian/status/1297857565385093121

He returned to Hong Kong from Spain and upon RT-PCR entry screening at the HK airport tested positive again by RT-PCR on Aug 15 (4.5 months after his initial positive test), with Ct=26.7, a fairly high viral load. No symptoms the second time. Hospitalized again. (2/6)

Viral load declined while hospitalized. Tested negative for IgG from hospitalization day 1-3, but seroconverted (became IgG+) on day 5. Sequencing of the virus from March and August saliva samples show two distinct #SARSCoV2 viruses from different clades (V/19A vs G/20A). (3/6)

Read 6 tweets

David R. Liu

@davidrliu

8 Jul 20

@nature

Today we report in @nature, in collaboration with the labs of Joseph Mougous and @VamsiMootha, the development of a new class of CRISPR-free base editors that enable precision editing of mitochondrial DNA (mtDNA) for the first time. drive.google.com/file/d/1xHcs__… 1/7

The team discovered DddA, an interbacterial toxin that is a novel cytidine deaminase enzyme that operates on *double-stranded* DNA (dsDNA), unlike all previously known cytidine deaminases, which require single-stranded DNA (ssDNA). 2/7

Previous efforts to precisely edit mtDNA have been stymied by the fact that CRISPR requires guide RNAs, which have not been effectively delivered into the mitochondria. mtDNA can be cut with ZFNs or TALENs, but doing so results in mtDNA destruction rather than precise editing.3/7

Read 7 tweets

David R. Liu

@davidrliu

8 May 20

A key piece of the #COVID19 #SARSCoV2 origin puzzle emerges: where did the virus's unusual spike protein RBD—the receptor-binding domain that engages human ACE2 protein and allows the virus to enter human cells—come from? A new study from Shen & coworkers provides an answer.(1/5)

The authors isolated coronaviruses from pangolins dying from respiratory disease in a wildlife rescue center in Mar-Aug 2019. One CoV-positive pangolin had antibodies that cross-reacted with #SARSCoV2 antigens, suggesting the animal was infected with a SARS-CoV-2-like virus.(2/5)

The pangolin #coronavirus was highly similar to #SARSCoV2 in several key proteins, including a remarkably similar spike protein RBD that differs from the SARS-CoV-2 RBD at only ONE amino acid position out of ~457 amino acids. (3/5)

Read 6 tweets

David R. Liu

@davidrliu

27 Apr 20

As some communities begin to lift #COVID19 stay-at-home orders, let’s talk about #masks. There is extensive evidence that masks protect you, and thus protect those around you. For example, this meta-analysis of 51 studies and 49 publications: bmj.com/content/bmj/33… (1/9)

But what type of mask should you wear? Any type of mask that serves as a barrier between your nose/mouth and the environment will likely help and is better than no mask, but data has been published on cloth masks vs surgical (medical) masks vs N95 masks (respirators). (2/9)

In the above meta-study, surgical masks reduce the average odds of being infected with #SARSCoV, the virus that causes SARS and a cousin of the #SARSCoV2 virus that causes #COVID19, by 68%—even better than frequent hand washing (55%)—of course, best is to do both. (3/9)

Read 9 tweets

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