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]

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.

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/