We are immensely grateful for the continued support from the Swedish Research Council (project: “CRISPR-based studies of pathogenic neutrophil biology”)

https://www.vr.se/inenglish/newspress/newsarchive/news2017/grantdecisionmedicineandhealth.5.557786f115f1aee2670eab19.html 

A hyper-accurate Cas9 variant from the Doudna lab!

Nature. 2017 Sep 20. doi: 10.1038/nature24268. [Epub ahead of print]

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.

Abstract

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1-4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown5-9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

PMID: 28931002

 

DOI: 10.1038/nature24268

CRISPR screen identifies that CMTM6 regulates the expression of PD-1L and anti-tumor immunity!

Nature. 2017 Aug 16. doi: 10.1038/nature23643. [Epub ahead of print]

CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity.

Burr ML1,2,3, Sparbier CE1, Chan YC1, Williamson JC3, Woods K4,5, Beavis PA1,2, Lam EYN1,2, Henderson MA1,2, Bell CC1,2, Stolzenburg S1, Gilan O1,2, Bloor S3, Noori T1, Morgens DW6, Bassik MC6, Neeson PJ1,2, Behren A4,5, Darcy PK1,2, Dawson SJ1,2,7, Voskoboinik I1,2, Trapani JA1,2, Cebon J4,5, Lehner PJ3, Dawson MA1,2,7,8.

Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR-Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.

 

Similar data was also published back to back by the Schumacher lab

–> https://www.ncbi.nlm.nih.gov/pubmed/28813410/