A great study from the Zhang lab developing and comparing different novel Cas9 variants

https://pubmed.ncbi.nlm.nih.gov/32187529 

Mol Cel. 2020 May 21;78(4):794-800.e8. Epub 2020 Mar 17.

Highly Parallel Profiling of Cas9 Variant Specificity

Jonathan L Schmid-Burgk, Linyi Gao, David Li, Zachary Gardner, Jonathan Strecker, Blake Lash, Feng Zhang

Abstract
Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.

Keywords: CRISPR-Cas9; Cas9 specificity variants; double-strand break detection; off-target analysis; tagmentation.

New plasmids from us deposited to AddGene

We have deposited two plasmids generated by drs. Sudeepta Kumar Panda and Sanjay Boddul, used in our recent PNAS paper (Panda et al, PNAS, 2020), to Addgene.

The first plasmid is a development of the LentiGuide-Puro from the Zhang lab where Sudeepta inserted a P2A-EGFP cassette enabling both Puro selection and EGFP based selection. We typically use the EGFP signal to evaluate the MOI and monitor the efficiency of puro based selection:

https://www.addgene.org/137729/

 

The second plasmid was developed from the LentiGuide-Puro-P2A-EGFP by Sanjay to also include an RFP reporter in the stuffer region driven by a bacterial promoter. This enables you to rapidly evaluate if bacteria expanded on agar/amp plates have taken up constructs where the stuffer is intact or not. This is very helpful to evaluate how well the cloning of a CRISPR/gRNA library, replacing the stuffer, into the plasmid has worked. A big thanks to the molecular biology wizard Erik Holmgren for help with the mRFP1 sequence.

https://www.addgene.org/137730/

Our neutrophil IL-4 study is finally out!

Our neutrophil IL-4 study is finally out! The study identifies several interesting regulatory effects by IL-4 with relevance for the autoimmune context and basic neutrophil biology.

I am also very happy starting to publish data from our CRISPR discovery platform that we use to rapidly describe biological processes and identify potential drug targets in the context of autoimmunity and cancer immunotherapy.

Great work everyone involved: Sudeepta, Gustaf, Long, Yanek, Vaish, Yunbing, Sanjay, André, Bruno, and Zsolt! 

Paper:

https://www.pnas.org/content/early/2020/01/23/1914186117/tab-article-info

Press release:

https://news.ki.se/proteins-that-protect-against-joint-inflammation-identified

Genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese)!

For the paper: https://www.biorxiv.org/content/early/2018/07/02/356626

For the plasmids: https://www.addgene.org/browse/article/25689/ 

 

Up, down, and out: optimized libraries for CRISPRa, CRISPRi, and CRISPR-knockout genetic screens

Kendall R SansonRuth E HannaMudra HegdeKatherine F DonovanChristine StrandMeagan E SullenderEmma W VaimbergAmy GoodaleDavid E RootFederica Piccioni, John G Doench

Abstract

Advances in CRISPR-Cas9 technology have enabled the flexible modulation of gene expression at large scale. In particular, the creation of genome-wide libraries for CRISPR knockout (CRISPRko), CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) has allowed gene function to be systematically interrogated. Here, we evaluate numerous CRISPRko libraries and show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we developed genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese). Negative selection screens showed that Dolcetto substantially outperforms existing CRISPRi libraries with fewer sgRNAs per gene and achieves comparable performance to CRISPRko in the detection of gold-standard essential genes. We also conducted positive selection CRISPRa screens and show that Calabrese outperforms the SAM library approach at detecting vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.

Great links for making sense of TCR nomenclature!

Mouse Vb6 is the same as TRBV19.  Human Vb3 is the same as TRBV28. It´s confusing, to say the least.

Here are some links for finding antibodies for different TCR genes!!:

Human TRAV:

http://www.imgt.org/IMGTrepertoire/Regulation/antibodies/human/TRA/TRAV/Hu_TRAVMab.html

Human TRBV:

http://www.imgt.org/IMGTrepertoire/Regulation/antibodies/human/TRB/TRBV/Hu_TRBVMab.html

Mouse TRAV:

http://www.imgt.org/IMGTrepertoire/index.php?section=Regulation&repertoire=antibodies&species=mouse&group=TRAV

Mouse TRBV:

http://www.imgt.org/IMGTrepertoire/index.php?section=Regulation&repertoire=antibodies&species=mouse&group=TRBV

For a bit more of an overview, as suggested to me by Dr. Daniel Pellicci, have a look at “Correspondence between nomenclatures” also at IMGT /  http://www.imgt.org/IMGTrepertoire/LocusGenes/#J