We have deposited our favorite CRISPR screen plasmid @Addgene

It’s a version of LentiGuide-puro where Sudeepta Panda and Long Jiang added P2A-eGFP after PuroR, lac-mRFP in the stuffer, and a cloning site directly after the sgRNA scaffold.

The plasmid allows for both puro and eGFP selection and MOI determination. The lac-mRFP in the stuffer gives a simple visual quality control step when expanding in bacteria (red=stuffer, white=spacer successfully introduced). The cloning site allows for introducing e.g. UMIs.


Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles

Beautiful paper from the Elling  and Zuber  Labs on a novel score to predict sgRNA efficiency. Also including a website for sgRNA design based on the VBC score: https://www.vbc-score.org/ . Brought to my attention by shared first author Julian Jude.

Paper: https://pubmed.ncbi.nlm.nih.gov/32514112/

Commentary: https://www.viennabiocenter.org/about/news/new-prediction-tool-sharpens-the-crispr-scissors/

Our review “Designing custom CRISPR libraries for hypothesis-driven drug target discovery” is online!

Here we describe the bioinformatic tools we use designing and analyzing hypothesis-driven CRISPR Screens.

Great work by talented PhD student Vaishnavi Iyer, and everyone else involved!

–> https://www.sciencedirect.com/science/article/pii/S2001037020303627?via%3Dihub

Interesting study by Orange et al – PRIME Cells Predicting Rheumatoid Arthritis Flares

N Engl. J. Med. 2020 Jul 16;383(3):218-228. doi: 10.1056/NEJMoa2004114. PMID: 32668112

RNA Identification of PRIME Cells Predicting Rheumatoid Arthritis Flares

Dana E OrangeVicky YaoKirsty SawickaJohn FakMayu O FrankSalina ParveenNathalie E BlachereCaryn HaleFan ZhangSoumya RaychaudhuriOlga G TroyanskayaRobert B Darnell



Background: Rheumatoid arthritis, like many inflammatory diseases, is characterized by episodes of quiescence and exacerbation (flares). The molecular events leading to flares are unknown.

Methods: We established a clinical and technical protocol for repeated home collection of blood in patients with rheumatoid arthritis to allow for longitudinal RNA sequencing (RNA-seq). Specimens were obtained from 364 time points during eight flares over a period of 4 years in our index patient, as well as from 235 time points during flares in three additional patients. We identified transcripts that were differentially expressed before flares and compared these with data from synovial single-cell RNA-seq. Flow cytometry and sorted-blood-cell RNA-seq in additional patients were used to validate the findings.

Results: Consistent changes were observed in blood transcriptional profiles 1 to 2 weeks before a rheumatoid arthritis flare. B-cell activation was followed by expansion of circulating CD45-CD31-PDPN+ preinflammatory mesenchymal, or PRIME, cells in the blood from patients with rheumatoid arthritis; these cells shared features of inflammatory synovial fibroblasts. Levels of circulating PRIME cells decreased during flares in all 4 patients, and flow cytometry and sorted-cell RNA-seq confirmed the presence of PRIME cells in 19 additional patients with rheumatoid arthritis.

Conclusions: Longitudinal genomic analysis of rheumatoid arthritis flares revealed PRIME cells in the blood during the period before a flare and suggested a model in which these cells become activated by B cells in the weeks before a flare and subsequently migrate out of the blood into the synovium. (Funded by the National Institutes of Health and others.).

A CRISPR-based safety switch for cell therapies published by the Porteus lab

–> https://pubmed.ncbi.nlm.nih.gov/32661439/
Nat. Biotechnol. 2020 Jul 13. doi: 10.1038/s41587-020-0580-6. PMID: 32661439

Metabolic engineering generates a transgene-free safety switch for cell therapy

Volker WiebkingJames O PattersonRenata MartinMonica K ChandaCiaran M LeeWaracharee SrifaGang BaoMatthew H Porteus



Safeguard mechanisms can ameliorate the potential risks associated with cell therapies but currently rely on the introduction of transgenes. This limits their application owing to immunogenicity or transgene silencing. We aimed to create a control mechanism for human cells that is not mediated by a transgene. Using genome editing methods, we disrupt uridine monophosphate synthetase (UMPS) in the pyrimidine de novo synthesis pathway in cell lines, pluripotent cells and primary human T cells. We show that this makes proliferation dependent on external uridine and enables us to control cell growth by modulating the uridine supply, both in vitro and in vivo after transplantation in xenograft models. Additionally, disrupting this pathway creates resistance to 5-fluoroorotic acid, which enables positive selection of UMPS-knockout cells. We envision that this approach will add an additional level of safety to cell therapies and therefore enable the development of approaches with higher risks, especially those that are intended for limited treatment durations.