This webpage accompanies our practical course on CRISPR/Cas gene targeting and nanopore DNA sequencing. Detailed information can be found in our publication. The course was originally developed by Hauke Holtorf, Bernd Dittrich, Thomas Dürr and Jürgen Braun. Röbbe Wünschiers and Robert Maximilian Leidenfrost expanded the course by nanopore DNA sequencing. The following figure illustrates the complete experiment.

In the laboratory, plasmid DNA is cut by the site specific Cas9 endonuclease. It finds its target with a guide RNA that is previously synthesised from a DNA template. Afterwards, the cut plasmid is sequenced in order to determine the precise cutting site. Although known by the teacher, it is to be determined by the students.

For DNA sequencing the MinION sequencer from Oxford Nanopore is employed. The following video show preparing the MinION equipped with a Flongle flow cell for sequencing.

Finally, the sequence reads produced by the MinION are mapped to the original plasmid and the result is visualised. These bioinformatic steps are executed by our server, accessible through this website. You just have to upload the Fast5 file produced by the sequencer. To test this data analysis pipeline, you can download a demo Fast5 file.

Take a look at this video to see our experiment in action (german only).

In 2012, Jennifer Doudna and Emmanuelle Charpentier co-authored a seminal article in the journal Science describing a programmable, RNA-guided endonuclease¹. This enzyme, known as Cas9, leads to targeted double-strand breaks in the DNA². When repaired by a billion-year-old cellular machinery, the cell can be tricked to insert specific nucleotides or complete genes. While the former allows, e.g., for the repair of genetic diseases, the latter can introduce new functions to cells. Already in 2020, both scientists were awarded the Nobel Prize for developing a genetic engineering tool from what was originally discovered as an adaptive bacterial immune system. One major advantage of this tool, named the CRISPR/Cas gene editing system, is its ease of use: Only a specific guide RNA and the Cas enzyme are required.

In 2018, the Chinese He Jiankui became the first scientist ever who gene-edited human fertilised egg cells³. After successful implantation and birth, two became known as Nana and Lulu – the first twins with a genetically manipulated germ line. Clearly, an ethical boundary was crossed and his experiment outlawed worldwide. Nonetheless, gene editing or, in a broader sense, genome editing does also hold promises to cure people from genetic diseases and to develop, e.g., crops that withstand pests and climatic challenges⁴.

However, the before said has not been possible without reading the DNA in an efficient and economical way. In the 1970s, Frederick Sanger developed a method to read the DNA sequence with the help of a DNA polymerase⁵. He received the Nobel Prize in 1980 for what became known as Sanger sequencing. Ever since, DNA sequencing was tedious lab work with devices costing tens of thousands of euros. Until 2014, when the UK-based company Oxford Nanopore Technologies released its first portable sequencing device that employs protein nanopores for DNA sequencing^6-7. Their device, named MinION, costs less that one thousand euros and fits into your palm.

Gaining insight into the effect of genetics by reading DNA and associating DNA sequences with phenotypes is the basis for DNA editing, e.g., with the CRISPR/Cas gene editing system. Both methods lay the foundation for future-oriented medicine, breeding, conservation biology or genetic engineering, to name but a few, and ultimately for a modern bioeconomy⁸.