Author: fredrikwermeling

Am I ready for CRISPR? A user’s guide to genetic screens by Dr. John G. Doench.

Highly recommended review regarding CRISPR screens:

https://www.nature.com/articles/nrg.2017.97.pdf

Abstract

Exciting new technologies are often self-limiting in their rollout, as access to state-of-the-art instrumentation or the need for years of hands-on experience, for better or worse, ensures slow adoption by the community. CRISPR technology, however, presents the opposite dilemma, where the simplicity of the system enabled the parallel development of many applications, improvements and derivatives, and new users are now presented with an almost paralyzing abundance of choices. This Review intends to guide users through the process of applying CRISPR technology to their biological problems of interest, especially in the context of discovering gene function at scale.

PMID: 29199283

iBiology – A fantastic series of biology videos. Highly recommended!

https://www.ibiology.org/biology-topics/

“iBiology’s mission is to bring the stories of cutting-edge research in the life sciences to a global audience without paywalls or other barriers to learning. Our primary means of communication is through videos, so the scientists themselves explain their research and the process of scientific discovery.

By providing a direct link to scientists, iBiology bridges the divides that have emerged between the lab bench and the classroom, and academia and the general public. We also provide special materials geared for educators and professional development for science trainees. While not all the content will be accessible to every audience level, all of our products are infused with the same spirit: that scientific progress is fueled by an open exchange of ideas. Thus this project reflects the ethos of science, as hundreds of researchers have donated their time and effort to share their insights and the excitement of discovery on this open forum.

iBiology.org was founded in 2006 by Ron Vale, a professor at University of California, San Francisco and a Howard Hughes Medical Institute Investigator. It has grown to include a wide variety of materials that now consists of hundreds of talks, lectures, interviews, courses, documentary storytelling, and other materials.”

Depmap (Broad Institute and collaborators) – Full genomic CRISPR screens >500 cancer cell lines (and counting).

https://depmap.org/portal/ 

Depmap – A Cancer Dependency Map to systematically identify genetic and pharmacologic dependencies and the biomarkers that predict them.

Cancer Dependency Map

The mutations that cause cancer cells to grow also confer specific vulnerabilities that normal cells lack. Some of these acquired alterations represent compelling therapeutic targets. The challenge is that, for the overwhelming majority of cancers, we do not fully understand the relationship between the genetic alterations of cancer and the dependencies they cause. To solve this problem, we are creating a “cancer dependency map” by systematically identifying genetic dependencies and small molecule sensitivities and discovering the biomarkers that predict them.

DepMap scientists are profiling hundreds of cancer cell line models for genomic information and sensitivity to genetic and small molecule perturbations. By triangulating information from these and other large-scale datasets, the hope is to define a landscape of genetic targets for therapeutic development, identify patients that who respond to these therapies, and develop a better understanding of the vulnerabilities of cancer.

The DepMap project at the Broad Institute is part of a strategic collaboration with theWellcome Sanger Institute (Hinxton, UK). By leveraging the expertise and infrastructure available at both organisations, we aim to more rapidly deliver a high-quality DepMap. We anticipate that this foundational dataset will catalyse a new wave of precision cancer medicines.

Dependency Map

I made a new video about how to look at FASTQ files from a CRISPR screen experiment using Excel (not recommended)

 

Using Excel is not recommended for analyzing your CRISPR screen data. I recommend to instead using CRISPRAnalyzer (http://crispr-analyzer.dkfz.de/). However, using Excel could be an easy way to get an overview of the result as the video hopefully shows.

The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.

CRISPR RNAs trigger innate immune responses in human cells.

Kim S, Koo T, Jee HG, Cho HY, Lee G, Lim DG, Shin HS, Kim JS.

Genome Res. 2018 Feb 22. doi: 10.1101/gr.231936.117. [Epub ahead of print]

PMID: 29472270

https://www.ncbi.nlm.nih.gov/pubmed/29472270 

 

Abstract

Here, we report that CRISPR guide RNAs (gRNAs) with a 5′-triphosphate group (5′-ppp gRNAs) produced via in vitro transcription triggerRNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5′-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5′-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4+ T cells. These results are in line with previous findings that chemically synthesized sgRNAs with a 5′-hydroxyl group are much more efficient than in vitro-transcribed (IVT) sgRNAs in human and other mammalian cells. The phosphatase treatment of IVT sgRNAs is a cost-effective method for making highly active sgRNAs, avoiding innate immune responses in human cells.