For many molecular biologists, the discovery of the CRISPR-Cas9 system marked a new milestone in research: finally, genomic DNA can be cut with high efficiency and precision, enabling genes to be disabled, modified or re-introduced.
This requires little more than a snippet of RNA from the genetic material, which takes the Cas9 protein scissors to the point in the DNA that is to be cut. This RNA snippet, known as sgRNA (single guide RNA), contains a sequence of 20 RNA-letters complementary to the genomic target site that scientists have hitherto had to select laboriously by hand or with a variety of online tools. In some cases, it was then uncertain whether the sgRNA would take the Cas9 gene scissors to the right place or to a similar but unwanted place in the genome and whether the sgRNA efficiency was high.
A new tool in the CRISPR-Cas9 toolbox
The new “CrispRGold” program written by PhD student Robin Graf from the MDC research group headed by Prof. Klaus Rajewsky makes it significantly easier to disable specific genes.
The program searches a defined DNA target sequence in order to identify the best place for the cut and suggests an sgRNA sequence that is unique in the genetic material and hence delivers the Cas9 protein only to the required point. Cas9 can then snip the gene so that it ceases to function. The algorithm is based on experimental data as well as the uniqueness and other properties of the sequences.
Test on hard-to-cultivate mouse cells
With his colleague Dr. Van Trung Chu, Graf tested the system on certain white blood cells in the mouse, the B cells. These cells cannot be cultivated for any length of time, because they do not survive long outside their natural environment. Genes must therefore be deactivated quickly and in as many cells as possible in order to study their function. Chu achieved this by breeding a genetically modified line of mice that produces large yet well-tolerated quantities of the Cas9 gene scissors. The researchers then isolated the B cells from these mice and delivered sgRNAs specific for individual genes to these cells. With high reproducibility, the sgRNAs designed with CrispRGold destroyed the target genes in on average 80 percent of the cells – “an excellent rate”, says Graf. “High efficiency and a low error rate are absolutely essential in low-throughput experiments of this sort.”
The researchers used their new method to identify a number of previously unknown genes that are involved in B-cell development. The CrispRGold program will now be made available online so that it can be used by scientists worldwide: “The program can easily be used for other types of cell from a wide range of organisms. It could also be relevant to clinical applications – it treats sequence uniqueness as a high priority and thus minimises the risk of potentially unwanted gene modifications, which must be avoided at all costs in gene therapy,” says Graf. CrispRGold is expected to be available atfrom November 2016.
Deoxyribonucleic acid (DNA) is a long-chain molecule that carries genetic information in the form of genes. The DNA chain normally exists in the form of a double helix. Ribonucleic acid (RNA) is also a chain-shaped carrier of genetic information; it is usually produced as the DNA is read. Unlike DNA, RNA can fold itself into complex loops and three-dimensional structures. CRISPR-RNA must have a particular structure in order to work correctly.
The CRISPR-Cas9 helps bacteria defend themselves against viruses. CRISPR stands for “Clustered Regularly Interspaced Short Palindromic Repeats”; CRISPRs are short repetitions of gene sequences in the genetic material of the bacterium. They contain copies of viral genetic material and they conduct Cas proteins (CRISPR-associated proteins) to the place in the virus DNA that needs to be destroyed. Molecular biologists have started to use the system as a tool in recent years because of its flexibility, efficiency and precision.
Anup Arumughan1, Yvette Roske1, Carolin Barth1, Laura Lleras Forero1, Kenny Bravo-Rodriguez4, Alexandra Redel1, Simona Kostova1, Erik Mcshane1, Robert Opitz1, Katja Faelber1, Kirstin Rau1, Thorsten Mielke2, Oliver Daumke1, Matthias Selbach1, Elsa Sanchez-Garcia4, Oliver Rocks1, Daniela Panáková1, Udo Heinemann1,3, Erich E. Wanker1 (2016): „Quantitative interaction mapping reveals an extended UBX domain in ASPL that disrupts functional p97 hexamers.“ Nature Communications.
1Max Delbrück Center for Molecular Medicine, Berlin, Germany; 2Max Planck Institute for Molecular Genetics, Berlin, Germany; 3Institute for Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany; 4Max-Planck-Institute for Coal Research, Mülheim an der Ruhr, Germany
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