For the fourth meeting in the two year-long project „European Challenges in Gene Editing by CRISPR“, the Erasmus+ team met at Vilniaus Jezuitu Gimnazia in Vilnius to the execution of a lab-workshop with pioneer character. For the first time on international level did students perform DNA-sequencing in a lab using a Third Generation method developed by Oxford Nanopore. The students and their teachers from France, Denmark, Germany, Greece, Poland, Slovakia and Czech Republic were warmly welcomed by the Lithuanian hosts. We were especially happy about the participation of the American delegation, consisting of students of the Governor’s School for Science and Mathematics, our longtime partner school from Hartsville (South Carolina).

From 27.09.2023 – 06.10.2023 the, in May 2023 in Istanbul developed, education unit on the topic of DNA-sequencing of a CRISPR/Cas9 cut in a plasmid was evaluated. Besides the practical execution of the experiment, education material was presented and thus put to the test.

The laboratory internship was instructed by an international mentor team, consisting of students from Poland, Denmark and Lithuania. During the first step of the multiple day-long lab internship the CRISPR guide RNA (gRNA), which is specifically programmable and leads the Endonuclease Cas9 to a specific target in the genome, was synthesized by all workgroups by the method of in-vitro transcription. The successful synthesis of the gRNA was verified by means of gel-electrophoresis. Thereon the workgroups brought the Endonuclease Cas9 together with the gRNA and thus formed the active CRISPR/Cas9-complex, the so-called gene scissor. Again, by gel-electrophoresis, the successful cut of the complex into the circular DNA of the plasmid pBR322 (4361 bp) and its following linearization was verified. Finally by the sequencing method we evaluated whether the cut was actually specifically or rather randomly made.

As the highlight of the workshop the linearized plasmid was sequenced via Oxford Nanopore technology, a Third Generation sequencing method. Supported by the sequencing results the precise cut of the Endonuclease CRISPR/Cas9 could be exactly proven by all of the 22 international workgroups. The laboratory internship was supported by the globally operating biotechnology company Thermo Fisher Scientific, which provided us the necessary lab tools and chemicals. Moreover, we were invited to the company to visit their research facilities.

Furthermore, a visit to Vilnius University was scheduled. The Erasmus+ team was invited by the renowned Lithuanian scientist Prof. Virginijus Šikšnys,a pioneer on the field of gene-editing by the means of CRISPR/Cas9. After his exciting Lecture he took extensive time to answer questions from students and teachers. The meeting was rounded off by the visit of the medieval Castle in Trakai, erected in the 15^th Century, and a wonderful final dinner at which we could reflect thr impressive experiences of this workshop.

At the end of the meeting all participants were on the same page that the shared development of educational material on CRISPR/Cas9 and sequencing were enabled thanks to the funding of the European Union and the pioneer work was to the benefit of all the international students.