Web-Tool Description Downloads Authors FAQ


Is there a publication explaining the CLUE pipeline in detail?

Yes, there is. We have published a paper in Nucleic Acids Research containing explanations, lots of data and a detailled protocol.
Find the link here: CLUE in Nucleic Acids Research

Do you provide examples for the different input file formats?

Yes, we have an example file for each of the two different formats. You can download them directly here:

Which sgRNA expression vectors are compatible with the Clue protocol?

The online tool allows you to choose from 2 different sgRNA expression vector systems. First, there are our own sgRNA expression vectors, which contain the H1 RNA Pol. III promoter and the new or improved sgRNA scaffold published by Chen et al., 2013, Cell. This is the vector system we have used to establish the entire pipeline. We are planning to deposit this vector on Addgene - until then, feel free to request it from us. Alternatively, you may choose vectors from the pLentiGuide (Addgene: #52963) or pLentiCRISPR (Addgene: #52961) families. These vectors use the U6 RNA Pol. III promoter.

What is the difference between the “old” and “new” sgRNA scaffold?

The difference between the two sgRNA scaffolds are just a handful of base changes. Importantly, the old sgRNA scaffold has a stretch of 4 Ts just after the sgRNA, which can act as a transcriptional terminator. The new sgRNA scaffold has a number of changes that circumvent this problem.

How are sgRNAs selected from the reference libraries? How are they prioritized?

Independent of the chosen CRISPR-variant, Clue always picks sgRNA starting from the top of the lists provided by the corresponding publications listed in Table S1 of the paper. For example, sgRNAs from the Wang et al. human CRISPRko library were ranked based on the predicted cleavage efficacy (see Wang et al., Science, 2015). Thus, sgRNAs are always selected based on their efficiency.

All gene names I enter end up in the “Genes not found” tabs. What went wrong?

Our tool works according to the official nomenclature of genes from Genenames.org (for human genes) and MGI (for murine genes) and thus is case sensitive. If you want to generate sgRNA libraries for human genes for example, the gene names should all be in capitals. Accordingly, if you want to enter mouse gene names only the first letter should be capitalized.

For some genes, I get twice as many sgRNAs as I asked for. Why is that?

This may be the case in sgRNA libraries for CRISPRi/a. Our reference libraries for these two applications contain sgRNAs for more than one transcript for some of the genes. If you request sgRNAs for such a gene, CRISRP Clue provides you with your requested number of sgRNAs for each of those transcripts.

How can I analyze the cloning efficiency of my sgRNA libraries after NGS?

We provide some more bioinformatic tools for these analyses in the Downloads section. These tools are written in Python and can be executed via the command line. We know that this may present a hurdle for some wet-lab scientists, but we have tried to make these tools as easy to use as possible by providing detailed descriptions on how to run them alongside the script itself.

Which sgRNA libraries are referenced by Clue when I enter gene lists?

The reference sgRNA libraries we have used are those published from the Sabatini, Weissmann and Doench labs. All CRISPRi/a sgRNAs originate from Horlbeck et al., 2016, eLife, while human k.o. sgRNAs are from Wang et al., 2015, Science and murine k.o. sgRNAs are from Doench et al., 2016, Nature Biotechnology. Please note that currently our murine k.o. reference library is the Brie library, which only contains 4 sgRNAs per gene.

In the wetlab cloning procedure, do I have to perform PCR 1 and TOPO cloning or could I directly start with PCR 2 and immediately clone the individual sgRNA libraries?

In principle it is possible to directly perform amplification of an sgRNA library from the oligo-pool without prior sub-cloning into the TOPO vector. However, often the initial amount of oligonucleotide pool DNA obtained from the vendor is a limiting factor. For example, if 100 ng of DNA are initially provided, but the oligopool contains more than 10 sgRNA libraries, it is not possible to run a PCR with 10 ng of template for each library. Furthermore, this procedure would not allow to repeat PCR reactions if something went wrong. In contrast, when the oligopool is first sub-cloned into the TOPO vector, the amount of template DNA for any of the sgRNA libraries is virtually infinite. Also, sequencing of the entire pool allows assessing the quality and distribution of the oligopool obtained. A disadvantage of the sub-cloning procedure is the fact that one more PCR, as well as one more cloning step is needed in the entire workflow.