We are very grateful for the continuous support from the Swedish Rheumatism Association and the Stiftelsen Professor Nanna Svartz Fond

The Swedish Rheumatism Association:

https://www.reumatikerforbundet.org/om-oss/about-swedish-rheumatism-association/

Stiftelsen Professor Nanna Svartz Fond:

http://www.stiftelseansokan.se/Pages/Svartz.aspx

TIDE –> Identification of Indel frequencies in a CRISPR targeted cell population

TIDE: Tracking of Indels by DEcomposition

TIDE is a web tool which rapidly assesses genome editing of a target locus by CRISPR-Cas9. Based on the quantitative sequence trace data from two standard capillary (Sanger) sequencing reactions, the TIDE software quantifies editing efficacy and identifies the predominant types of insertions and deletions (indels) in the DNA of a targeted cell pool. See Brinkman et al. 2014 Nucl. Acids Res.for a detailed explanation and examples.

https://tide.deskgen.com/

The Broad Cancer Cell Line Encyclopedia!

A great resource find gene expression patterns in human cancer cell lines:

https://portals.broadinstitute.org/ccle

Intresting CRISPR tool from Microsoft Research CRISPR

https://crispr.ml/

Combinatorial genetic screens –> The Doench lab presents “Big Papi”!

Yet another impressive study from the Doench lab. https://www.ncbi.nlm.nih.gov/pubmed/29251726/

2017 Dec 18. doi: 10.1038/nbt.4048. [Epub ahead of print]

Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.

Najm FJ^1,^2,³, Strand C¹, Donovan KF¹, Hegde M¹, Sanson KR¹, Vaimberg EW¹, Sullender ME¹, Hartenian E¹, Kalani Z¹, Fusi N⁴, Listgarten J⁴, Younger ST¹, Bernstein BE^1,^2,³, Root DE¹, Doench JG¹.

Abstract

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The “Big Papi” (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.

PMID: 29251726
DOI: 10.1038/nbt.4048

We´re enormously grateful for the continued support from the Åke Wiberg Foundation (project “Regulating the activity of antibodies in health and disease”)!

–> http://ake-wiberg.se/

Trim-Away – an interesting approach to degrade proteins in cells using antibodies and the Fc-binding protein TRIM21

2017 Nov 13. pii: S0092-8674(17)31255-2. doi: 10.1016/j.cell.2017.10.033. [Epub ahead of print]

A Method for the Acute and Rapid Degradation of Endogenous Proteins.

Clift D¹, McEwan WA², Labzin LI², Konieczny V³, Mogessie B³, James LC⁴, Schuh M⁵.

Author information

Abstract

Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.